Recombinant host cells and methods for the production of l-lactic acid

ABSTRACT

Provided herein are recombinant fungal or bacterial cells comprising a heterologous L-lactate dehydrogenase, and processes of preparing L-lactic acid employing such cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. 119(e)and Article 2 of the Paris Convention for the Protection of IndustrialProperty (1883) to U.S. Provisional Application No. 62/657,432, filedApr. 13, 2018, U.S. Provisional Application No. 62/809,156, filed Feb.22, 2019, and U.S. Provisional Application No. 62/809,196, filed Feb.22, 2019, the content of which are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted herewith viaEFS-web in computer readable form, which is hereby incorporated byreference in its entirety for all purposes. The ASCII copy, created onMar. 4, 2019, is named LYGOS_0024_01_WO_ST25 and is 68 KB in size.

FIELD

This disclosure relates to methods and materials for the production ofL-lactic acid including, for example, isolated synthetic and naturalnucleic acids, synthetic and natural polypeptides, host cells, andmethods and materials for producing L-lactic acid by direct fermentationfrom carbon sources, along with methods of preparing L-lactic acidpolymers.

BACKGROUND

Lactic acid has been used historically for food acidulation andpreservation. Recently, the global market for lactic acid has increaseddue to demand for biodegradable plastics in food packaging, medicaldevices, and personal care products, all of which comprise lacticacid-based polymers such as polylactic acid (PLA), which ispredominantly manufactured from L-lactic acid. PLA can be a competitivereplacement for petrochemical-derived plastics that employ depletingfeedstocks and hazardous, energy-intensive manufacturing processes.

Current PLA polymers are used for single-use products, such asdisposable, non-microwaveable food packaging. However, high-temperaturePLA can replace the durable petrochemical plastics polypropylene andpolystyrene, which opens up new market applications.

L-lactic acid can be manufactured by microbial fermentation. However,existing materials and methods to produce high purity L-lactic acid bymicrobial fermentation are incapable of satisfying performance metricsat commercial scale (see, for example, Okano et al, Appl MicrobiolBiotechnol (2010) 85:413-423). Existing bacterial fermentations may alsorequire complex and expensive nutrients in fermentation media,prohibiting applications at commercial scale. In the yeast Saccharomycescerevisiae, efforts may be impaired by the inability of engineered hostcells to grow, which lead to low L-lactic acid yields andproductivities. There is a need to produce L-lactic acid in high yield,in some instances with acid tolerant organisms and with reducedbyproduct formation from bio-based, renewable sources.

SUMMARY

The long-term economic and environmental concerns associated with thepetrochemical industry have provided the impetus for the development anduse of renewable chemicals (such as bio-based chemicals) that can beutilized instead of petroleum-derived chemicals. Such renewablechemicals include lactic acids, which are important building blockchemicals that are used in a wide range of industries and applications,including polypropylene and polystyrene. Recent development ofbiorefining processes which convert renewable feedstocks into bio-basedlactic acid can provide the necessary reagents for producing bio-basedproducts. As a more sustainable alternative to petrochemically-derivedproducts, there is a great need for bio-based L-lactic acid and polymersmade therefrom, such as bio-based polypropylene and polystyrene, as wellas methods of making these renewable compositions.

The present disclosure provides materials and methods for efficientproduction of high purity and high yield L-lactic acid by microbialfermentation. These recombinant cells have alterations to one or moreof: (1) L-lactic acid pathway enzyme(s), (2) ancillary proteins gene(s)and (3) byproduct pathway enzyme(s). The present disclosure furtherdescribes methods for purifying and analyzing fermentation productsynthesized by such recombinant cells and L-lactic acid-based polymers.

In one aspect, the present disclosure provides a recombinant cellcomprising a heterologous nucleic acid encoding L-lactate dehydrogenase.In some embodiments, the L-lactate dehydrogenase has at least 60% aminoacid identity with SEQ ID NO: 1. In some embodiments, the heterologousnucleic acid is expressed in sufficient amount to produce L-lactic acid.In some embodiments, the recombinant cell is a yeast cell. In someembodiments, the recombinant cell is a prokaryotic cell.

In some embodiments, the present disclosure provides a recombinant cellfurther comprising one or more heterologous nucleic acids encoding oneor more proteins selected from organic acid transporters and redoxcofactor biogenesis proteins. In some embodiments, the heterologousnucleic acid encodes an organic acid transporter having at least 60%amino acid identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 5. In some embodiments, the present disclosure provides arecombinant cell further comprising a genetic disruption of one or moregenes encoding a pyruvate decarboxylase, a protein subunit of thepyruvate dehydrogenase complex, glycerol-3-phosphate dehydrogenase,NAD(P)H dehydrogenase, or combinations thereof.

In another aspect, provided herein is a method for producing L-lacticacid that comprises culturing the recombinant cells of this disclosureunder fermentation conditions suitable to produce L-lactic acid, or asalt thereof. In some embodiments, the method further comprisesisolating the L-lactic acid, or salt thereof.

In another aspect, provided here is a method for producing a lactic acidpolymer that comprises culturing the recombinant cell of this disclosureunder fermentation conditions suitable to produce L-lactic acid, or asalt thereof. In some embodiments, the method comprises isolating theL-lactic acid or salt thereof. In some embodiments, the method comprisesoptionally converting the L-lactic acid or salt thereof to an L-lacticacid derivative. In some embodiments, the method comprises producing alactic acid polymer using the isolated L-lactic acid, the salt thereof,or the L-lactic acid derivative.

DETAILED DESCRIPTION

The present disclosure provides materials and methods, and embodimentsfor the biological production and purification of L-lactic acid. ThisDetailed Description contains parts identified by headings merely for areader's convenience, and, as will be apparent to the skilled artisan,disclosure found in any part can be relevant to any other part of thisdisclosure. The present disclosure is not limited to particular nucleicacids, expression vectors, enzymes, biosynthetic pathways, hostmicroorganisms, processes, or enantiomers, as such may vary. Becauselactic acid encompasses two different enantiomers—D-lactic acid(synonymous with R-lactic acid and (+)-lactic acid) and L-lactic acid(synonymous with S-lactic acid and (−)-lactic acid)—many materials,methods, and embodiments disclosed that relate to L-lactic acid alsopertain to D-lactic acid. In addition, many modifications may be made toadapt a particular situation, material, composition of matter, process,process steps or process flows, in accordance with this disclosure. Allsuch modifications are within the scope of the claims appended hereto.

Definitions

As used herein, the following terms have the following meanings.

The term “heterologous” as used herein refers to a material that isnon-native to a cell. In one embodiment, a nucleic acid is heterologousto a cell, and so is a “heterologous nucleic acid” with respect to thatcell, if at least one of the following is true: 1) the nucleic acid isnot naturally found in that cell (that is, it is an “exogenous” nucleicacid); 2) the nucleic acid is naturally found in a given host cell (thatis, “endogenous to”), but the nucleic acid or the RNA or proteinresulting from transcription and translation of this nucleic acid isproduced or present in the host cell in an unnatural (for example,greater or lesser than naturally present) amount; 3) the nucleic acidcomprises a nucleotide sequence that encodes a protein endogenous to ahost cell but differs in sequence from the endogenous nucleotidesequence that encodes that same protein (having the same orsubstantially the same amino acid sequence), typically resulting in theprotein being produced in a greater amount in the cell, or in the caseof an enzyme, producing a mutant version possessing altered (forexample, higher or lower or different) activity; and/or 4) the nucleicacid comprises two or more nucleotide sequences that are not found inthe same relationship to each other in the cell. In an embodiment, aprotein is heterologous to a host cell if it is produced by translationof RNA or the corresponding RNA is produced by transcription of aheterologous nucleic acid. In another embodiment, a protein is alsoheterologous to a host cell if it is a mutated version of an endogenousprotein, and the mutation was introduced by genetic engineering.

The terms “homologous,” “homology,” “identity,” “sequence identity” andvariations thereof refer to the similarity of a nucleic acid or aminoacid sequence, in some embodiments in the context of a coding sequencefor a gene or the amino acid sequence of a protein. Homology or identitysearches can be employed using a known, or reference, amino acid orcoding sequence for a useful protein to identify coding sequences orproteins that have similar sequences and thus are likely to perform thesame function as the protein defined by the reference sequence. Aprotein having homology or high sequence identity to a reference proteincan be identified, for example and without limitation, by a BLAST(https://blast.ncbi.nlm.nih.gov) search. A protein with high percenthomology or sequence identity is highly likely to carry out theidentical biochemical reaction as the reference protein. In anembodiment, two enzymes having greater than 60% homology or sequenceidentity will carry out identical biochemical reactions, and the higherthe homology or sequence identity, i.e., 65%, 70%, 75%, 80%, 85%, 90% orgreater than 95% homology or sequence identity, the more likely the twoproteins have the same or similar function. In another embodiment, aprotein with at least 60% homology or sequence identity to its referenceprotein is defined as homologous to its reference protein.

Generally, homologous proteins share substantial sequence identity. Setsof homologous proteins generally possess one or more specific aminoacids that are conserved across all members of the consensus sequenceprotein class. The percent sequence identity of a protein relative to aconsensus sequence is determined by aligning the protein sequenceagainst the consensus sequence. Various sequence alignment algorithmsare suitable for aligning a protein with a consensus sequence. See, forexample, Needleman, S B, et al., “A general method applicable to thesearch for similarities in the amino acid sequence of two proteins.”Journal of Molecular Biology 48 (3): 443-53 (1970). Following alignmentof the protein sequence relative to the consensus sequence, thepercentage of positions where the protein possesses an amino aciddescribed by the same position in the consensus sequence determines thepercent sequence identity or homology to the consensus sequence. When adegenerate amino acid is present in a consensus sequence, any of theamino acids described by the degenerate amino acid may be present in theprotein at the aligned position for the protein to be identical to theconsensus sequence at the aligned position. In one embodiment, when itis not possible to distinguish between two closely related amino acids,the following one-letter symbols may be used—“B” refers to aspartic acidor asparagine; “Z” refers to glutamine or glutamic acid; “J” refers toleucine or isoleucine; and “X” or “+” refers to any amino acid. A dash(-) in a consensus sequence indicates that there is no amino acid at thespecified position.

In addition to identification of useful enzymes by percent homology orsequence identity with a given consensus sequence, in one embodimentenzymes useful in the compositions and methods provided herein can alsobe identified by the number of highly conserved amino acid residuesrelative to a consensus sequence. For the consensus sequence providedherein, a number of highly conserved amino acid residues are described.In this embodiment, enzymes useful in the compositions and methodsprovided herein have a substantial number, and sometimes all, of thehighly conserved amino acids at positions aligning with the indicatedresidues in the consensus sequence. As with percent homology or sequenceidentity, the presence or absence of these highly conserved amino acidscan be determined by alignment of the query protein sequence relative tothe consensus sequence, as described above.

The terms “expression vector” or “vector” refer to a nucleic acid and/ora composition comprising a nucleic acid that can be introduced into ahost cell, for example, by transduction, transformation, or infection,such that the cell then produces (i.e., expresses) nucleic acids and/orproteins contained in or encoded by the sequence of the vector, which insome embodiments are nucleic acids and/or proteins other than thosenative to the cell, or in a manner not native to the cell. Thus, an“expression vector” contains nucleic acids to be expressed by the hostcell. Optionally, the expression vector can be contained in materials toaid in achieving entry of the nucleic acids into the host cell, such asthe materials associated with a virus, liposome, protein coating, or thelike. Expression vectors suitable for use in various aspects andembodiments of the present disclosure comprise those into which anucleic acid sequence can be, or has been, inserted, along with anyoperational elements. Thus, an expression vector can be transferred intoa host cell and replicated therein. In an embodiment, an expressionvector that integrates into chromosomal, mitochondrial, or plastid DNAis employed. In another embodiment, an expression vector that replicatesextrachromosomally is employed. Typical expression vectors includeplasmids, and expression vectors typically contain operational elementsfor transcription of a nucleic acid in the vector.

The terms “ferment”, “fermentative”, and “fermentation” are used hereinto describe culturing microbes under conditions to produce usefulchemicals, including but not limited to conditions under which microbialgrowth, be it aerobic or anaerobic, occurs.

The terms “host cell”, “recombinant host cell,” “recombinant cell” and“recombinant host microorganism” are used interchangeably herein torefer to a living cell that can be, or has been, transformed viainsertion of an expression vector. A host cell or microorganism asdescribed herein may be a prokaryotic cell (for example, a microorganismof the kingdom Eubacteria) or a eukaryotic cell. A prokaryotic celllacks a membrane-bound nucleus, while a eukaryotic cell has amembrane-bound nucleus.

The terms “isolated” or “pure” refer to material that is substantially,for example, greater than 50%, 75%, 90%, 95%, 98% or 99%, free ofcomponents that normally accompany it in its native state, for example,the state in which it is naturally found or the state in which it existswhen it is first produced. Additionally, any reference to a “purified”material is intended to refer to an isolated or pure material.

The terms “genetic disruption,” “genetic modification” and “geneticalteration” are used interchangeably to refer to ways of alteringgenomic, chromosomal or plasmid-based gene expression. Non-limitingexamples of genetic disruptions include gene editing (for exampleCRISPR/Cas9, zinc finger nucleases, TALEN), RNAi, nucleic aciddeletions, nucleic acid insertions, nucleic acid substitutions, nucleicacid mutations, knockouts, premature stop codons, transcriptionalpromoter modifications, and the like. Genetic disruptions give rise toaltered gene expression and or altered protein activity. Altered geneexpression encompasses decreased, eliminated and increased geneexpression levels. In one embodiment, altered gene expression results inaltered protein expression.

As used herein, “recombinant” refers to the alteration of geneticmaterial by human intervention. In some embodiments, recombinant refersto the manipulation of DNA or RNA in a cell or virus or expressionvector by molecular biology/recombinant DNA technology methods, forexample cloning and recombination. Recombinant can also refer tomanipulation of DNA or RNA in a cell or virus by random or directedmutagenesis. A “recombinant” cell or nucleic acid can be described withreference to how it differs from a naturally occurring, wild-typecounterpart. In this disclosure, reference to a cell or nucleic acidthat has been “engineered” or “modified” and variations of those terms,refers to a recombinant cell or nucleic acid.

The terms “transduce”, “transform”, “transfect”, and variations thereofrefer to the introduction of one or more nucleic acids into a cell. Inan embodiment, the nucleic acid is stably maintained or replicated bythe cell for a sufficient period of time to enable the function(s) orproduct(s) it encodes to be expressed. Stable maintenance or replicationof a nucleic acid may take place either by incorporation of the sequenceof nucleic acids into the cellular chromosomal DNA, for example thegenome as occurs by chromosomal integration, or by replicationextrachromosomally, as occurs with a freely-replicating plasmid. A viruscan be stably maintained or replicated when it is “infective”: when ittransduces a host microorganism, replicates, and spreads progenyexpression vectors, for example, viruses, of the same type as theoriginal transducing expression vector to other microorganisms.

“L-lactic acid” means the dextrorotatory molecule having the chemicalformula C₃H₆O₃ and a molecular mass of 90.078 g/mol (CAS No. 79-33-4).The terms “S-lactic acid”, “(+)-lactic acid”, “L-(+)-lactic acid”,“(2S)-2-hydroxypropanoic acid”, “(S)-(+)-lactic acid”,“(S)-2-hydroxypropanoic acid”, “(S)-2-hydroxypropionic acid”,“(S)-lactic acid”, “(S)-α-hydroxypropionic acid” all describe the samemolecule and are used interchangeably in the art.

In conditions with pH values higher than the pKa of L-lactic acid (forexample, about pH>3.86 when using a base, such as sodium hydroxide),L-lactic acid is deprotonated to the L-lactate anion C₃H₅O₃ ⁻. In thisdisclosure, “L-lactate anion” is used interchangeably with “L-lactate”,“(+)-lactate”, “S-(+)-lactate”, “(2S)-2-hydroxypropanoate”,“(S)-2-hydroxypropanoate”, “(S)-2-hydroxypropionate”, “(S)-lactate”,“(S)-α-hydroxypropionate.”

The L-lactate anion is capable of forming an ionic bond with a cation toproduce an L-lactate salt. In this disclosure, the term “L-lactate”refers to a variety of L-lactate salt forms and is used interchangeablywith “L-lactate salts”. Non-limiting examples of L-lactates comprisesodium L-lactate, calcium L-lactate, and lithium L-lactate. L-lactatesalts can crystallize in various states of hydration.

The L-lactic acid, L-lactate salt, and L-lactate esters, of the presentdisclosure are synthesized from biologically produced organic componentsby a fermenting microorganism. For example, L-lactic acid, L-lactatesalts, or their precursor(s) are synthesized from the fermentation ofsugars by recombinant host cells of the present disclosure.

The prefix “bio-” or the adjective “bio-based” may be used todistinguish these biologically-produced L-lactic acid and L-lactatesalts from those that are derived from petroleum feedstocks. The terms“bio-based” or “non-petrochemically derived” or “renewable” as usedherein refer to an organic compound that is synthesized frombiologically produced organic components by fermenting a microorganism.For example, an acid which was itself synthesized from glucose (forexample, derived from cornstarch) by a genetically engineeredmicroorganism is bio-based or non-petrochemically derived. As usedherein, a compound of renewable or non-petrochemical origin comprisescarbon atoms that have a non-petrochemical origin. These compounds aredistinguished from wholly petroleum-derived compounds or those entirelyof fossil origin. Such compounds have a ¹⁴C amount substantially higherthan zero, such as about 1 parts per trillion or more, because they arederived from photosynthesis-based starting material, such as forexample, glucose or another feedstock used in producing such a compound.

The redox cofactor nicotinamide adenine dinucleotide, NAD, comes in twoforms—phosphorylated and un-phosphorylated. The term “NAD(P)” or “NADP”refer to both phosphorylated (NADP) and un-phosphorylated (NAD) formsand encompasses oxidized versions (NAD+ and NADP+) and reduced versions(NADH and NADPH) of both forms. The term “NAD(P)+” refers to theoxidized versions of phosphorylated and un-phosphorylated NAD, i.e.,NAD+ and NADP+. Similarly, the term “NAD(P)H” refers to the reducedversions of phosphorylated and un-phosphorylated NAD, i.e., NADH andNADPH. When NAD(P)H is used to describe the redox cofactor in an enzymecatalyzed reaction, it indicates that NADH and/or NADPH is used.Similarly, when NAD(P)+ is the notation used, it indicates that NAD+and/or NADP+ is used. While many proteins may bind either aphosphorylated or un-phosphorylated cofactor, there are redox cofactorpromiscuous proteins, natural or engineered, that are indiscriminate; inthese cases, the protein may use either NADH and/or NADPH. In someembodiments, enzymes that preferentially utilize either NAD(P) or NADmay carry out the same catalytic reaction when bound to either form.

Various values for temperatures, titers, yields, oxygen uptake rate(OUR), and pH are recited in the description and in the claims. In someembodiments, these values are not exact and can be approximated to therightmost/last/least significant FIGURE. For example, a temperaturerange of from about 30° C. to about 42° C. covers the range 25° C. to44° C.

D-lactic acid and L-lactic acid are enantiomers (also known as opticalisomers); they are molecules that share the same molecular weight of90.078 g/mol and are non-superimposable mirror images of each other,analogous to one's left and right hands being the same and notsuperimposable by simple reorientation around an axis. The D- or(+)-enantiomer rotates polarized light clockwise (to the right) whilethe L- or (−)-enantiomer rotates polarized light counterclockwise (tothe left). Solutions with a mixture of both enantiomers are racemicmixtures and are typically produced by microbes that naturally producelactic acid. Lactic acid enantiomers are prohibitively expensive toseparate out at commercial scale. High enantiomeric purity (i.e., 99.5%and above) enables one to titrate specific physical properties of PLAblends; enantiomer-exclusive precursors, catalysts and/or enzymes willtypically be present for a biosynthetic pathway to give rise to anenantiomerically/optically pure product (i.e., a lactic acid solutionthat comprises only one of the two enantiomers at >99.5% purity).

In a one aspect, this disclosure provides recombinant host cells capableof producing L-lactic acid comprising one or more heterologous nucleicacids that encode the L-lactic acid biosynthetic pathway, wherein thepathway enzymes comprise an L-lactate dehydrogenase (LLDH).

In some embodiments, the recombinant host cells comprise one or moreheterologous nucleic acids encoding the LLDH from Lactobacillushelveticus. In another embodiment, the recombinant host cells providedherein overexpress two copies of Lactobacillus helveticus L-lactatedehydrogenase (LDH) expressed from a constitutive TDH1 promoter. Incertain embodiments, if an LDH is not overexpressed, no (or only traceamounts) of lactic acid are produced. In some embodiments, therecombinant host cells comprise heterologous nucleic acids encoding anL-lactate dehydrogenase with at least 60% amino acid sequence homologyto SEQ ID NO: 1.

In some embodiments, the L-lactate dehydrogenase comprises the residuesGXGXXG, where X refers to any amino acid, and wherein 1-3 amino acidresidues located 20-22 residues downstream to the GXGXXG sequence ismutated, in some embodiments to a neutral amino acid, as compared to awild type sequence.

In some embodiments, the L-lactate dehydrogenase comprises the residuesGXGXXG, where X refers to any amino acid, and wherein a negativelycharged amino acid, such as D, 20-22 located residues downstream to theGXGXXG residue is changed to a neutral amino acid, as compared to a wildtype sequence.

In some embodiments, the recombinant host cell is a yeast cell. Incertain embodiments, the yeast cell belongs to the Issatchenkiaorientalis/Pichia fermentans clade. In some embodiments, the yeast cellbelongs to the genus Pichia, Issatchenkia or Candida. In someembodiments, the yeast cell is Pichia kudriavzevii. In some embodiments,the yeast cell belongs to the Saccharomyces clade. In some embodiments,the yeast cell is Saccharomyces cerevisiae. In other embodiments, therecombinant host cell is a prokaryotic cell. In some embodiments, theprokaryotic cell belongs to the genus Escherichia, Corynebacterium,Bacillus, or Lactococcus. In some embodiments, the prokaryotic cell isEscherichia coli, Corynebacterium glutamicum, Bacillus subtilis, orLactococcus lactis.

In another aspect, the disclosure provides recombinant host cells thatfurther comprise one or more heterologous nucleic acids encoding one ormore ancillary proteins that function in redox cofactor biogenesisand/or organic acid transport. In some embodiments, the one or moreancillary proteins comprise a protein with at least 60% amino acidsequence homology with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, or any combination thereof.

In another aspect, the disclosure provides recombinant host cells thatfurther comprise a genetic disruption of one or more genes wherein theone or more genes encode pyruvate decarboxylase, a protein subunit ofthe pyruvate dehydrogenase complex, glycerol-3-phosphate dehydrogenase,NAD(P)H dehydrogenase, or any combination thereof. In some embodiments,the one or more genes has at least 60% sequence homology to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11 or any combination thereof. In some of these embodiments, therecombinant host cells produce less than 5 g/L of ethanol, less than 5g/L of pyruvate, less than 5 g/L of acetate, less than 5 g/L ofglycerol, or any combination thereof, in the fermentation broth.

In another aspect, the disclosure provides a method for the productionof L-lactic acid that comprises culturing the recombinant host cells ofthe disclosure for a sufficient period of time to produce L-lactic acid.In some embodiments, the method further comprises an oxygen transferrate greater than 5 mmol/L/hr. In some embodiments, the method furthercomprises an operational temperature of between about 25° C. and about45° C. In some embodiments, the method further comprises a finalfermentation broth pH of between pH 2-5. In some embodiments, the methodfurther comprises providing at least 100 g/L glucose to the recombinanthost cell and producing an L-lactic acid yield of at least 25%. In someembodiments, the method further comprises production of L-lactic acidwith an enantiomeric purity of at least 99.5%.

In another aspect, the disclosure provides a method for the recovery ofL-lactic acid and L-lactate salts from the fermentation broth.

The recombinant host cells, and processes provided herein areadvantageous because they utilize aerobic fermentation. First, glucose,which is a carbon source for producing L-lactic acid, is catabolizedfaster under aerobic conditions than under anaerobic conditions. Second,a small fraction of glucose catabolized under aerobic conditions is usedfor ATP production, thereby enabling higher L-lactic acid titers andproductivity, while allowing the fermentation to be run at a low pH. Aseries of genetic modifications are used, as provided herein, to enableefficient L-lactic acid production in an aerobic fermentation.

In one embodiment, the recombinant host cells provided and utilizedherein comprise URA2 gene knockouts. Accordingly, such host cells areuracil auxotrophic strains. The uracil auxotrophic strains utilizedherein are advantageous for various reasons. First, the auxotrophyallows for controlling the amount of biomass formed by changing theamount of uracil supplemented in the fermentation medium. Second, uponexhaustion of uracil, the strain no longer grows but still consumesglucose at a fast rate. Overall, this enables a two-phase fermentation,where depending on the availability of uracil, growth is separated fromlactic acid production. When uracil is available, the strain grows. Whenuracil is depleted, the strain stops growing but efficiently convertsglucose to lactic acid.

In another embodiment, the recombinant host cells provided hereincomprise a knockout of one or more, in some embodiments two or more, inother embodiments three, pyruvate decarboxylase (PDC) genes selectedfrom PDC1, PDC5, and PDC6. Pyruvate decarboxylases are responsible forconversion of pyruvate to acetaldehyde; accordingly, pyruvatedecarboxylase knockouts reduce formation of acetaldehyde-derivedproducts and increases lactic acid production by increasing pyruvatesubstrate availability for LDH in the cytosol. In another embodiment,the recombinant host cells provided herein overexpress an engineered PDCgene (for example, one or more of PDC1, PDC5, or PDC6) that contains atemperature-sensitive intein. At low temperature (for example, at about30° C.) the PDC protein is active, whereas at higher temperatures (forexample, at about 37-42° C.) the PDC protein is no longer functionallyexpressed and the host cells exhibit reduced pyruvate decarboxylaseactivity, and eventually increased lactic acid production.

In another embodiment, the recombinant host cells provided hereincomprise a knockout of mitochondrial external NADH dehydrogenase (EC1.6.99.3), which is NDE1. In another embodiment, the recombinant hostcells provided herein comprise a knockout of mitochondrial external NADHdehydrogenase, which is NDE2. In another embodiment, the recombinanthost cells provided herein comprise a knockout of NDE1, and NDE2. NADHdehydrogenase is responsible for a majority of NADH re-oxidationactivity in the cytosol under aerobic conditions. The knockout enablesefficient lactic acid production under aerobic conditions bysubstantially eliminating a major source of native NADH re-oxidationactivity in the cytosol. Thus, LDH activity becomes the major route viawhich the cell re-oxidizes NADH under aerobic conditions in the absenceof growth.

In another embodiment, the recombinant host cells provided hereinoverexpress an engineered NADH dehydrogenase gene (for example, NED1and/or NED2) that contains a temperature-sensitive intein. At lowtemperature (for example, at about 30° C.) the NADH dehydrogenaseprotein is active, whereas at higher temperatures (for example, at about37-42° C.) the NADH dehydrogenase protein is no longer functionallyexpressed and the host cells exhibit reduced NADH dehydrogenaseactivity, and eventually increased lactic acid production.

In another embodiment, the recombinant host cells provided hereincomprise a knockout of a PDA1 gene, which is a subunit of pyruvatedehydrogenase. Pyruvate dehydrogenase is responsible for convertingpyruvate to acetyl-CoA in the mitochondria. The knockout reducesformation of acetyl-CoA derived products and increases lactic acidproduction by increasing pyruvate substrate availability for LDH in thecytosol.

In another embodiment, the recombinant host cells provided hereinoverexpress an engineered PDA1 gene that contains atemperature-sensitive intein. At low temperature (for example, at about30° C.) the PDA1 protein is active and it enables good conversion ofpyruvate to acetyl-CoA in the mitochondria. The acetyl-CoA is used forgrowth, ATP formation, and, more general cellular activities. At highertemperatures (for example, at about 37-42° C.) the PDA1 protein is nolonger functionally expressed and the host cells exhibit reduced PDA1activity. In this manner, a two-phase fermentation with respect totemperature is enabled. Low temperature is thus beneficial for growthand high temperature for lactic acid production. Therefore, in certainembodiments, temperature and uracil are used to efficiently switch fromgrowth to lactic acid production phase.

In another embodiment, the recombinant host cells provided hereincomprise a ZWF1 knockout. ZWF1 is a cytoplasmic glucose-6-phosphatedehydrogenase (EC 1.1.1.49). These knockouts decrease carbon fluxthrough the pentose phosphate pathway, thereby decreasing NADPHproduction and increasing NADH production during the conversion ofglucose to pyruvate via glycolytic enzymes. The increased amount of NADHmay increase lactic acid production (yield, titer, and/or productivity)and decrease pyruvate accumulation. In another embodiment, therecombinant host cells provided herein overexpress an engineered ZWF1gene that contains a temperature-sensitive intein. At low temperature(for example, at about 30° C.) the ZWF1 protein is active and it enablespyruvate accumulation, and subsequently conversion of pyruvate toacetyl-CoA in the mitochondria. At higher temperatures (for example, atabout 37-42° C.) the ZWF1 protein is no longer functionally expressedand the host cells exhibit reduced ZWF1 activity, and eventuallyincreased lactic acid production.

In another embodiment, the recombinant host cells provided hereincomprise glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), GPD1knockouts. These knockouts decrease glycerol formation. Glycerol is amajor byproduct, which reduces lactic acid production; additionally,this knockout may increase the amount of NADH available for lactic acidproduction, resulting in increased lactic acid production and decreasedpyruvate accumulation. In another embodiment, the recombinant host cellsprovided herein overexpress an engineered GPD1 gene that contains atemperature-sensitive intein. At low temperature (for example, at about30° C.) the GPD1 protein is active, whereas at higher temperatures (forexample, at about 37-42° C.) the GPD 1 protein is no longer functionallyexpressed and the host cells exhibit reduced GPD 1 activity, andeventually increased lactic acid production.

In another embodiment, the recombinant host cells provided hereincomprise an NAD kinase (NADK; EC 2.7.1.23) knockout. In one embodiment,the NADK knockout is a YEF1 knockout. In another embodiment, the NADKknockout is a POSS knockout. These knockouts may decrease oxidation ofcytosolic NADH and NADPH, thereby increasing the cytosolic availabilityof NAD(P)H for re-oxidation by the LDH and may decrease pyruvateaccumulation and increase lactate production. In another embodiment, therecombinant host cells provided herein overexpress an engineered NADKgene (for example, a YEF1 and/or a POSS) that contains atemperature-sensitive intein. At low temperature (for example, at about30° C.) the NADK protein is active, whereas at higher temperatures (forexample, at about 37-42° C.) the NADK protein is no longer functionallyexpressed and the host cells exhibit reduced NADK activity, andeventually increased lactic acid production.

In another embodiment, the recombinant host cells provided hereincomprise a dihydrolipoamide dehydrogenase, DLD1B, knockout. A DLD1Bknockout decreases native lactic acid catabolism and may increaselactate production. In another embodiment, the recombinant host cellsprovided herein overexpress an engineered DLD1B gene that contains atemperature-sensitive intein. At low temperature (for example, at about30° C.) the DLD1B protein is active, whereas at higher temperatures (forexample, at about 37-42° C.) the DLD1B protein is no longer functionallyexpressed and the host cells exhibit reduced DLD1B activity, andeventually increased lactic acid production.

In one embodiment, if the host cell is a yeast cell, then the host cellexcludes: a deletion or disruption of at least one native L- orD-lactate:ferricytochrome c oxidoreductase gene. In another embodiment,if the host cell is yeast, the yeast strain produces more than about 80%g lactic acid per g glucose.

In another embodiment, if the host cell is of genera Candida, the hostcell does not express a metabolic phenotype that is a Crabtree-negativephenotype. In another embodiment, if the host cell expresses a metabolicphenotype that is a Crabtree-negative phenotype, the host cell is not ofgenera Candida. In another embodiment, if the host cell is of generaCandida, and the lactate dehydrogenase protein-encoding nucleic acid isoperatively linked to a promoter functional in a yeast cell from aCandida species that is a Candida sonorensis, Candida parapsilosis,Candida naeodendra, Candida methanosorbosa, Candida entomophila, Candidakrusei, Candida blankii, or Candida diddensiae cell, the host cell doesnot express a metabolic phenotype that is the Crabtree-negativephenotype.

In yet another aspect, provided herein is a method for producing alactic acid polymer comprising: culturing the recombinant host cells asdisclosed herein under fermentation conditions for a sufficient periodof time to produce L-lactic acid, or a salt thereof; optionallyconverting the L-lactic acid or salt thereof to a D-lactic acidderivative; recovering at least one of L-lactic acid, a salt thereof, ora derivative thereof, from the fermentation broth; and producing alactic acid polymer using the recovered L-lactic acid, a salt thereof,or a derivative thereof as at least one polymerization material.

Methods for converting L-lactic acid to poly L-lactic acid (PLLA), areknown in the art. Similarly, practitioners in the art are equipped touse and/or modify methods to convert L-lactic and D-lactic acid topoly(-D,L-lactide), and subsequently, a blended PLA, with varying D- toL-lactic acid ratios towards desired chemical and physical properties.

The L-lactic acid provided herein has non-petrochemical based carbons orhas a ¹⁴C amount substantially higher than zero, such as about 1 partsper trillion or more. Such non-petrochemical based (or bio-based orrenewable) L-lactic acid has a ¹⁴C amount substantially higher thanzero, such as about 1 parts per trillion or more, because they arederived from photosynthesis based starting material.

Recombinant Host Cells for Production of L-Lactic Acid

In certain embodiments, the present disclosure provides recombinant hostcells engineered to produce L-lactic acid, wherein the recombinant hostcells comprise one or more heterologous nucleic acids encoding one ormore L-lactic acid pathway enzymes. In particular embodiments, therecombinant host cells further comprise disruptions or deletions ofendogenous nucleic acids that improve yields, titers and/orproductivities of L-lactic acid. In some embodiments, the recombinanthost cells are capable of producing L-lactic acid under aerobicconditions. In some embodiments, the recombinant host cells are capableof producing L-lactic acid under substantially anaerobic conditions. Therecombinant host cells produce L-lactic acid at increased titers, yieldsand productivities as compared to a parental host cell that does notcomprise said heterologous nucleic acids.

In some embodiments, the recombinant host cells further comprise one ormore heterologous nucleic acids encoding one or more ancillary geneproducts (i.e., gene products other than the product pathway enzymes)that improve yields, titers and/or productivities of L-lactic acid. Inparticular embodiments, the recombinant host cells further comprisedisruptions or deletions of endogenous nucleic acids that improveyields, titers and/or productivities of L-lactic acid. In someembodiments, the recombinant host cells are capable of producingL-lactic acid under aerobic conditions. In some embodiments, therecombinant host cells are capable of producing L-lactic acid undersubstantially anaerobic conditions. The recombinant host cells produceone or more ancillary gene products at increased titers, yields andproductivities as compared to a parental host cell that does notcomprise said heterologous nucleic acids.

Any suitable host cell may be used in practice of the methods of thepresent disclosure, and in some examples, host cells useful in thecompositions and methods provided herein comprise archaeal, prokaryotic,or eukaryotic cells. In an embodiment of the present disclosure, therecombinant host cell is a prokaryotic cell. In an embodiment of thepresent disclosure, the recombinant host cell is a eukaryotic cell.

In an embodiment of the present disclosure, the recombinant host cell isa yeast cell. In various embodiments, yeast cells useful in the methodsof the present disclosure comprise yeasts of the genera Aciculoconidium,Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia,Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida,Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces,Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus,Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces,Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea,Holtermannia, Hormoascus, Hyphopichia, Issatchenkia, Kloeckera,Kloeckeraspora, Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces,Leucosporidium, Lipomyces, Lodderomyces, Malassezia, Metschnikowia,Mrakia, Myxozyma, Nadsonia, Nakazawaea, Nematospora, Ogataea,Oosporidium, Pachysolen, Phachytichospora, Phaffia, Pichia,Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes,Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora,Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus,Sporobolomyces, Sporopachydermia, Stephanoascus, Sterigmatomyces,Sterigmatosporidium, Symbiotaphrina, Sympodiomyces, Sympodiomycopsis,Torulaspora, Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea,Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis,Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis, andZygozyma, among others.

In various embodiments, the yeast cell is of a species selected from thenon-limiting group comprising Candida albicans, Candida ethanolica,Candida guilliermondii, Candida krusei, Candida hpolytica, Candidamethanosorbosa, Candida sonorensis, Candida tropicalis, Candida utilis,Cryptococcus curvatus, Hansenula polymorpha, Issatchenkia orientalis,Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromycesthermotolerans, Komagataella pastoris, Lipomyces starkeyi, Pichiaangusta, Pichia deserticola, Pichia galeiformis, Pichia kodamae, Pichiakudriavzevii (P. kudriavzevii), Pichia membranaefaciens, Pichiamethanolica, Pichia pastoris, Pichia salicaria, Pichia stipitis, Pichiathermotolerans, Pichia trehalophila, Rhodosporidium toruloides,Rhodotorula glutinis, Rhodotorula graminis, Saccharomyces bayanus,Saccharomyces boulardi, Saccharomyces cerevisiae (S. cerevisiae),Saccharomyces kluyveri, Schizosaccharomyces pombe (S. pombe) andYarrowia hpolytica.

The Crabtree phenomenon refers to the capability of yeast cells toconvert glucose to alcohol in the presence of high sugar concentrationsand oxygen instead of producing biomass via the tricarboxylic acid (TCA)cycle. Yeast cells produce alcohol to prevent growth of competingmicroorganisms in high sugar environments, which yeast cells can utilizelater on when the sugars are depleted. Many yeast can typically use twopathways to produce ATP from sugars: the first involves the conversionof sugars (via pyruvate) to carbon dioxide via the TCA cycle, and thesecond involves the conversion of sugars (via pyruvate) to ethanol.Yeast cells that display a Crabtree effect (known as Crabtree-positiveyeast cells) are able to simultaneously use both pathways. Yeast cellsthat do not display a Crabtree effect (known as Crabtree-negative yeastcells) only convert pyruvate to ethanol when oxygen is absent. In someembodiments of the present disclosure, the host cell is aCrabtree-positive yeast cell. In other embodiments, the host cell is aCrabtree-negative yeast cell. In certain embodiments, the host celldisplays a phenotype along a continuum of traits betweenCrabtree-positive and Crabtree-negative and is thus neither exclusivelya Crabtree-positive yeast cell nor Crabtree negative yeast cell. It isadvantageous to use a Crabtree-negative yeast or a yeast withperceptible Crabtree-negative tendencies or traits to produce L-lacticacid because high glucose concentrations can be maintained duringproduct biosynthesis without ethanol accumulation; ethanol is anundesired byproduct in L-lactic acid production. P. kudriavzevii doesnot produce appreciable amounts of ethanol from pyruvate at high glucoseconcentrations in the presence of oxygen, and as such is aCrabtree-negative yeast. In some embodiments, the host cell is P.kudriavzevii.

In certain embodiments, the recombinant yeast cells provided herein areengineered by the introduction of one or more genetic modifications(including, for example, heterologous nucleic acids encoding enzymesand/or the disruption of native nucleic acids encoding enzymes) into aCrabtree-negative yeast cell. In certain of these embodiments, the hostcell belongs to the Pichia/Issatchenkia/Saturnispora/Dekkera clade. Incertain of these embodiments, the host cell belongs to the genusselected from the group comprising Pichia, Issatchenkia, or Candida. Incertain embodiments, the host cell belongs to the genus Pichia, and insome of these embodiments the host cell is P. kudriavzevii. Members ofthe Pichia/Issatchenkia/Saturnispora/Dekkera or the Saccharomyces cladeare identified by analysis of their 26S ribosomal DNA using the methodsdescribed by Kurtzman C. P., and Robnett C. J., (“Identification andPhylogeny of Ascomycetous Yeasts from Analysis of Nuclear Large Subunit(26S) Ribosomal DNA Partial Sequences”, Atonie van Leeuwenhoek73(4):331-371; 1998). Kurtzman and Robnett report analysis ofapproximately 500 ascomycetous yeasts were analyzed for the extent ofdivergence in the variable D1/D2 domain of the large subunit (26S)ribosomal DNA. Host cells encompassed by a clade exhibit greatersequence identity in the D1/D2 domain of the 26S ribosomal subunit DNAto other host cells within the clade as compared to host cells outsidethe clade. Therefore, host cells that are members of a clade (forexample, the Pichia/Issatchenkia/Saturnispora/Dekkera or Saccharomycesclades) can be identified in one way using the methods of Kurtzman andRobnett.

In certain embodiments of the present disclosure, the recombinant hostcells are engineered by introduction of one or more geneticmodifications into a Crabtree-positive yeast cell. In certain of theseembodiments, the host cell belongs to the Saccharomyces clade. Incertain of these embodiments, the host cell belongs to a genus selectedfrom the group comprising Saccharomyces, Schizosaccharomyces,Brettanomyces, Torulopsis, Nematospora and Nadsonia. In certainembodiments, the host cell belongs to the genus Saccharomyces, and inone of these embodiments the host cell is S. cerevisiae.

In addition to yeast cells, other eukaryotic cells are also suitable foruse in accordance with methods of the present disclosure, so long as theengineered host cell is capable of growth and/or product formation.Illustrative examples of eukaryotic host cells provided by the presentdisclosure include, but are not limited to, cells belonging to thegenera Aspergillus, Crypthecodinium, Cunninghamella, Entomophthora,Mortierella, Mucor, Neurospora, Pythium, Schizochytrium,Thraustochytrium, Trichoderma, and Xanthophyllomyces. Examples ofeukaryotic strains include, but are not limited to, Aspergillus niger,Aspergillus oryzae, Crypthecodinium cohnii, Cunninghamella japonica,Entomophthora coronata, Mortierella alpina, Mucor circinelloides,Neurospora crassa, Pythium ultimum, Schizochytrium limacinum,Thraustochytrium aureum, Trichoderma reesei and Xanthophyllomycesdendrorhous.

Archaeal cells are also suitable for use in accordance with methods ofthe present disclosure, and in an embodiment of the present disclosure,the recombinant host cell is an archaeal cell. Illustrative examples ofrecombinant archaea host cells provided by the present disclosureinclude, but are not limited to, cells belonging to the genera:Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium,Pyrococcus, Sulfolobus, and Thermoplasma. Examples of archaea strainsinclude, but are not limited to Archaeoglobus fulgidus, Halobacteriumsp., Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Thermoplasma acidophilum, Thermoplasma volcanium, Pyrococcus horikoshii,Pyrococcus abyssi, and Aeropyrum pernix.

In an embodiment of the present disclosure, the recombinant host cell isa prokaryotic cell. Prokaryotic cells are suitable host cells forconstruction of recombinant metabolic pathways comprising heterologousenzymes catalyzing production of small-molecule products. Illustrativeexamples of recombinant prokaryotic host cells include, but are notlimited to, cells belonging to the genera Agrobacterium,Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter,Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium,Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus,Mesorhizobium, Methylobacterium, Microbacterium, Pantoea, Phormidium,Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus,Salmonella, Scenedesmun, Serratia, Shigella, Staphylococcus,Strepromyces, Synnecoccus, and Zymomonas. Examples of prokaryoticstrains include, but are not limited to, Bacillus subtilis (B.subtilis), Brevibacterium ammoniagenes, Bacillus amyloliquefacines,Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridiumacetobutylicum, Clostridium beigerinckii, Corynebacterium glutamicum (C.glutamicum), Enterobacter sakazakii, Escherichia coli (E. coli),Lactobacillus acidophilus, Lactococcus lactis, Mesorhizobium loti,Pantoea ananatis (P. ananatis), Pseudomonas aeruginosa, Pseudomonasmevalonii, Pseudomonas pudita, Rhodobacter capsulatus, Rhodobactersphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonellatyphi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, and Staphylococcus aureus.

Lactic Acid Pathway Enzymes

Provided herein in certain embodiments are recombinant host cells havingat least one active lactic acid pathway from pyruvate to lactate.Recombinant host cells having an active lactic acid pathway as usedherein are capable of producing one or more active enzymes capable ofcatalyzing the metabolic reaction in a lactic acid pathway, andtherefore are capable of producing lactic acid in measurable yieldsand/or titers when cultured under suitable conditions. Recombinant hostcells having a lactic acid pathway comprise one or more heterologousnucleic acids encoding lactic acid pathway enzyme(s) and are capable ofproducing lactic acid.

Eleven enzymatic steps are used to produce lactic acid from glucose. Thefirst 10 steps are the endogenous glycolysis pathway that convertsglucose to pyruvate. The last and 11th step uses a heterologous lactatedehydrogenase (LDH) enzyme to convert pyruvate to lactic acid. Alleleven enzymatic steps take place in the cytosol. The lactic acidpathway described herein produces lactate from glucose with thefollowing balanced, stoichiometric equation:

D-Glucose+2 ADP+2 orthophosphate=2 lactate+2 ATP+2H₂O

Recombinant host cells may employ combinations of metabolic reactionsfor biosynthetically producing the compounds of the present disclosure.The biosynthesized compounds produced by the recombinant host cellscomprise lactate, lactic acid, and the intermediates, products and/orderivatives of the lactic acid pathway. The biosynthesized compounds canbe produced intracellularly and/or secreted into the fermentationmedium.

L-Lactate Dehydrogenase

The lactic acid pathway comprises a lactate dehydrogenase (LDH) thatconverts one molecule of pyruvate and one molecule of reduced cofactorto one molecule of lactate and one molecule of oxidized cofactor. Invarious embodiments of the present disclosure, recombinant host cellscomprise one or more heterologous nucleic acids encoding a LDH, whereinsaid recombinant host cells are capable of producing lactic acid. Insome embodiments, the recombinant host cells comprise one or moreheterologous nucleic acids encoding one, more, or all of theaforementioned LDHs. In many embodiments, the LDH is derived from aprokaryotic source. In many embodiments, the LDH is derived from aeukaryotic source. Any enzyme is suitable for use in accordance withthis disclosure so long as the enzyme is capable of catalyzing said LDHreaction.

In the L-lactic acid pathway specifically, an NADH(P)H-dependent LLDH(EC #1.1.1.27) converts one molecule of pyruvate and one molecule ofreduced cofactor (for example, NAD(P)H) to one molecule of L-lactate andone molecule of oxidized cofactor (for example, NAD(P)+).

Most known LLDHs utilize NADH as the cofactor, and NADH-dependent LLDHwill generally be used when NADH is produced during the recombinant hostcell's glycolytic processes in converting glucose to pyruvate. In P.kudriavzevii and S. cerevisiae, for example, the glyceraldehyde3-phosphate dehydrogenase (GAPDH) in glycolysis reduces NAD+ to NADH;therefore, in embodiments wherein the GAPDH is NADH-producing, the LLDHis NADH-dependent.

Similarly, in embodiments wherein NADPH is the cofactor produced inglycolysis, the LLDH is NADPH-dependent. Kluyveromyces lactis andClostridium acetobutylicum, for example, natively express NADP+ reducingGAPDH enzymes, thereby generating NADPH in glycolysis; thus, whenengineering L-lactic acid production in these strain backgrounds, aNADPH-dependent LLDH used is NADPH-dependent. In other embodiments, theLLDH is engineered to preferentially utilize either NADH or NADPH ascofactors. In yet other embodiments, the LLDH is engineered to utilizedboth NADH and NADPH as cofactors.

A host cell can be engineered to produce a specific redox cofactor (NADHor NADPH) during glycolysis by changing the GAPDH enzyme expressed.Furthermore, both NADH and NADPH can be generated during glycolysisthrough concomitant expression of both NADH- and NADPH-dependent GAPDHenzymes. Lastly, NADPH and NADH can be interconverted through expressionof a transhydrogenase that catalyzes the interconversion of NADPH andNADH.

In various embodiments of the present disclosure, recombinant host cellscomprise one or more heterologous nucleic acids encoding a LLDH whereinsaid recombinant host cells are capable of producing L-lactic acid. Invarious embodiments of the present disclosure, recombinant host cellscomprise one or more heterologous nucleic acids encoding a LLDH whereinsaid recombinant host cells are capable of producing L-lactic acid. Insome embodiments, the recombinant host cells comprise one or moreheterologous nucleic acids encoding one, more, or all of the LLDHs froma genera selected from Lactobacillus, Bacillus, and Pediococcus. In someembodiments, the recombinant host cells comprise one or moreheterologous nucleic acids encoding one, more, or all of the LLDHsselected from Lactobacillus helveticus, Lactobacillus casei,Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus jensenii,Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillusfermentum, Lactobacillus acidophilus, Lactobacillus johnsonii, Bacillusmegaterium, Bacillus stearothermophylus, Bacillus Taurus, andPediococcus acidilactici. In another embodiment, the LLDH is fromLactobacillus helveticus.

In many embodiments, the LLDH is derived from a prokaryotic source. Inmany embodiments, the LLDH is derived from a eukaryotic source. Anyenzyme is suitable for use in accordance with this disclosure so long asthe enzyme is capable of catalyzing said LLDH reaction.

In certain embodiments, recombinant host cells comprise one or moreheterologous nucleic acids encoding one or more of the aforementionedL-lactic acid pathway, wherein the heterologous nucleic acids areexpressed in sufficient amounts to produce L-lactate. In variousembodiments, recombinant host cells may comprise multiple copies of asingle heterologous nucleic acid and/or multiple copies of two or moreheterologous nucleic acids. Recombinant host cells comprising multipleheterologous nucleic acids may comprise any number of heterologousnucleic acids.

The present disclosure also provides consensus sequences useful inidentifying and/or constructing the L-lactic acid pathway suitable foruse in accordance with the methods of the present disclosure. In variousembodiments, these consensus sequences comprise active site amino acidresidues which may contribute to substrate recognition and reactioncatalysis, as described below. Thus, an enzyme encompassed by aconsensus sequence provided herein has an enzymatic activity that isidentical, or essentially identical, or at least substantially similarwith respect to ability to catalyze the reaction performed by one of theenzymes exemplified herein. For example, a LLDH encompassed by a LLDHconsensus sequence provided herein has an enzymatic activity that isidentical, or essentially identical, or at least substantially similarwith respect to ability to convert one molecule of pyruvate and onemolecule of reduced cofactor to one molecule of L-lactate and onemolecule of oxidized cofactor (for example, NAD(P)+). As noted above,any protein substantially homologous to LLDH as described herein can beused in a host cell of the present disclosure. Further, any protein thatshares the specific function of LLDH as described herein can be used ina host cell of the disclosure despite comprising insufficient sequenceidentity with the LLDH consensus sequence.

In many embodiments, the LLDH is derived from a bacterial source. Inmany of these embodiments, the LLDH is derived from a host cellbelonging to a genus selected from the group comprising Aquifex,Bacillus, Enterococcus, Escherichia, Eubacterium, Fusobacterium,Klebsiella, Lactobacillus, Leuconostoc, Mycoplasma, Neisseria,Oenococcus, Pediococcus, Pseudomonas, Rhodopseudomonas, Selenomonas,Sporolactobacillus, Staphylococcus, Streptococcus, Thermodesulfatator,and Weisella.

In some embodiments, the LLDH is a Lactobacillus helveticus LLDH (aabv.LhLDH; UniProt ID: 032765; SEQ ID NO: 1) from Lactobacillus helveticusas shown below:

        10         20         30         40MAREEKPRKV ILVGDGAVGS TFAFSMVQQG IAEELGIIDI        50         60         70         80AKEHVEGDAI DLADATPWTS PKNIYAADYP DCKDADLVVI        90        100        110        120TAGAPQKPGE TRLDLVNKNL KILSSIVEPV VESGFEGIFL       130        140        150        160VVANPVDILT HATWRMSGFP KDRVIGSGTS LDTGRLQKVI       170        180        190        200GKMENVDPSS VNAYMLGEHG DTEFPAWSYN NVAGVKVADW       210        220        230        240VKAHNMPESK LEDIHQEVKD MAYDIINKKG ATFYGIGTAS       250        260        270        280AMIAKAILND EHRVLPLSVP MDGEYGLHDL HIGTPAVVGR       290 300               310        320KGLEQVIEMP LSDKEQELMT ASADQLKKVM DKAFKETGVK VRQ

In wild type LLDH, the residues GXGXXG, where X refers to any aminoacid, followed by a negatively charged amino acid 20-22 residuesdownstream is conserved. With respect to SEQ ID NO: 1, the conservedresidues are shown by underlines:

(SEQ ID NO: 1, aa 1-48) 1-MAREEKPRKVILVGDGAVGSTFAFSMVQQGIAEELGIIDIAKEHVEGD.

In certain embodiments, the LLDH provided and/or utilized herein ismutated as follows. In one embodiment, the mutation comprises changingD39 of SEQ ID NO: 1 (or another negatively charged residue such as D,which is 20-22 residues downstream from the conserved GXGXXG residue ofanother LLDH) to S/T/A/V/I/L/ or M.

In some embodiments, the mutation comprises changing one or both of thetwo residues following the conserved D39 residue of SEQ ID NO: 1. Insome embodiments, the mutation comprises changing one or both of the tworesidues following the conserved negatively charged residue, such as D,which negatively charged sequence is 20-22 residues downstream from theconserved GXGXXG residue of an LLDH.

In one embodiment, the mutation comprises changing 140 toR/H/S/T/A/V/K/L/ or M of SEQ ID NO: 1. In one embodiment, the mutationcomprises changing A41 to S/T/K/R/H/V/I/L/ or M of SEQ ID NO: 1. In someembodiment, the mutation comprises, the combination of two or more ofD39 to S/T/A/V/I/L/ or M; 140 to R/H/S/T/AN/K/L/ or M; and A41 toS/T/K/R/H/V/I/L/ or M of SEQ ID NO: 1.

In one embodiment, the LLDH mutant comprises the mutation D to S,wherein the D is 20-22 residues downstream from the conserved GXGXXGresidue of an LLDH. Methods of mutating a protein sequence are wellknown in the art and disclosed herein below. In certain embodiments,positions other than 39, 40, and 41 are conserved in the LLDH employedherein of SEQ ID NO: 1.

In one embodiment, the LLDH mutant comprises one or more of themutations D39S, I40R, and A41T of SEQ ID NO: 1. In one embodiment, theLLDH mutant comprises the mutations D39S, I40R, and A41T of SEQ IDNO: 1. In one embodiment, the LLDH mutant comprises the mutation I40R ofSEQ ID NO: 1. In one embodiment, the LLDH mutant comprises the mutationsD39S of SEQ ID NO: 1.

In certain embodiments, the LLDH employed in accordance with the presentdisclosure demonstrate increased activity overall, and increased k_(cat)and/or decreased K_(m) for NAD(P)H. In certain embodiments, the LLDHemployed in accordance with the present disclosure demonstrate increasedk_(cat) and/or decreased K_(m) for NADH. Overexpressing these enzymesresults in increased L-lactic acid production and decreased pyruvateaccumulation.

In many embodiments, recombinant host cells comprise one or moreheterologous nucleic acids encoding a LLDH wherein said recombinant hostcells are capable of producing L-lactic acid. In various embodiments,proteins suitable for use in accordance with methods of the presentdisclosure have LLDH activity and comprise an amino acid sequence withat least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95% sequence identity with SEQ ID NO: 1.

In many embodiments, the LLDH is derived from an archaeal source. Inmany of these embodiments, the LLDH is derived from a host cellbelonging to a genus selected from the group comprising Aeropyrum.

In many embodiments, the LLDH is derived from a eukaryotic source. Inmany of these embodiments, the LLDH is derived from a host cellbelonging to a genus selected from the group comprising Allomyces,Arabidopsis, Cardium, Haliotis, Helix, Limulus, Octopus, Phytophthora,Polysphondylium, Pythium, Rattus, and Saccharomyces.

Identifying and/or Improving L-Lactic Acid Pathway Enzymes

The following methods are useful as examples of methods for mutagenesisand diversification of genes for engineering specific or enhancedproperties of targeted enzymes. Practitioners in the art will appreciatethat the methods disclosed may be adapted as needed depending on thetarget enzyme properties desired. In some instances, the disclosedmethods are suitable for use in engineering enzymes towards improvedLLDH activity of the L-lactic acid pathway. In some embodiments, theLLDH is derived from an enzyme with native activity towards a substratethat is structurally similar to pyruvate.

Methods described herein comprise protein mutagenesis, identification,expression, purification, and characterization. Further, identificationof mutated proteins can comprise activity screens and phenotypicselections.

Generating Protein Libraries Via Mutagenesis

Enzymes that are identified as good mutagenesis starting points enterthe protein engineering cycle, which comprises protein mutagenesis,protein identification, protein expression, protein characterization,recombinant host cell characterization, and any combination thereof.Iterative rounds of protein engineering are typically performed toproduce an enzyme variant with properties that are different from thetemplate/original protein. Examples of enzyme characteristics that areimproved and/or altered by protein engineering comprise, for example,substrate binding (K_(m); i.e., a measure of enzyme binding affinity fora particular substrate) that includes non-natural substrateselectivity/specificity; enzymatic reaction rates (k_(cat); the turnoverrate of a particular enzyme-substrate complex into product and enzyme),to achieve desired pathway flux; temperature stability, for hightemperature processing; pH stability, for processing in extreme pHranges; substrate or product tolerance, to enable high product titers;removal of inhibition by products, substrates or intermediates;expression levels, to increase protein yields and overall pathway flux;oxygen stability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen. In some embodiments, the enzyme variantenables improved L-lactic acid pathway flux. In some embodiments, theenzyme variant enables increased L-lactate yield, titer and/orproductivity. In some embodiments, the enzyme variant enables increasedsubstrate specificity. In some embodiments, the enzyme variant displaysimproved kinetic properties, such as decreased K_(m) and/or increasedk_(cat). In some embodiments, the enzyme variant has increased K_(m)and/or decreased k_(cat) for the substrate pyruvate. In someembodiments, the enzyme variant has K_(m)<3 mM with pyruvate assubstrate. In some embodiments, the enzyme variant has k_(cat)>10turnovers per second with pyruvate as substrate. In some embodiments,the enzyme variant is a product of one or more protein engineeringcycles. In some embodiments, the enzyme variant comprises one or morepoint mutations.

In general, random and rational mutagenesis approaches are acceptablemethods for generating DNA libraries of mutant proteins. Error-prone PCRis a random mutagenesis method widely used for generating diversity inprotein engineering, and error-prone PCR is not only fast and easy, butit is also a method that has successfully produced mutated enzymes withaltered activity from a wild type DNA template. (Wilson, D. S. & Keefe,A. D. Random mutagenesis by PCR. Curr. Protoc. Mol. Biol. Chapter 8,Unit 8.3 (2001), To help increase the odds of identifying an enzyme with3-PG/2-PG phosphatase activity, rational mutagenesis of a small numberof active site mutations is also useful. Structural modeling allows oneto identify amino acids in the active site believed to be involved withsubstrate recognition. Other mutagenesis approaches that could be usedcomprise DNA shuffling and combinatorial mutagenesis. In someembodiments, the mutagenesis step is carried out more than once,resulting in iterative rounds of engineering.

Generating Strain Libraries Via Directed Evolution

In another aspect of this disclosure, directed evolution methods can beused to identify enzymes with LLDH activity and/or improved kineticparameters (for example, decreasing the enzyme Km and/or increasing theenzyme kcat when using pyruvate as the substrate) of enzymes exhibitingsuboptimal activity toward pyruvate. Directed evolution approaches areuseful in generating strain libraries with a wide diversity of mutationswherein the mutations are driven by the process of natural selectiongiven the constraints provided to the organism in its growthenvironment. Evolution approaches provide an effective and impartial wayof introducing sequence mutations that give rise to functional change atan organism scale, enabling practitioners to explore non-intuitivemutations in the universe of possibilities that lie beyond the confinesof one's understanding about structure-function specificity.

In some embodiments, a screen is designed to monitor the progress ofevolution over time. In some of these embodiments, it is useful to linkdesired mutagenesis with a measurable phenotype so that the rate ofevolution can be monitored over an extended period of time. In some ofthese embodiments, the measurable phenotype comprises cell growth,glucose consumption, and metabolite production. In some embodiments, themeasurable phenotype is favored by a selection. In some embodiments, thedirected evolution experiment is designed so that mutations acquired inthe target gene(s) is a measurable phenotype that is advantageous to theorganism. In some of these embodiments, the advantageous measurablephenotype comprises cellular fitness, energy production, growth rate,tolerance to toxicity, and tolerance to extreme culture conditions (suchas high or low pH, high or low temperature, high or low osmoticpressure, drought, and nutrient limitation). In various embodiments, oneor more synthetic metabolic pathways are constructed by introducingexogenous nucleic acids to recombinant host cells. In these embodiments,the one or more synthetic metabolic pathways provide a method ofapplying selective pressure or a method of selecting strain variantsthat result from directed evolution.

Besides a well-crafted screen and/or selection, before the evolutionexperiment begins, starting nucleic acid templates for proteins ofinterest (i.e., target gene(s) or parent gene(s)) can also beidentified. In embodiments of the present disclosure, enzymes that serveas a good starting point for LLDH engineering are identified. In theseembodiments, LLDH-encoding nucleic acids are integrated into the genomeof recombinant host cells. In some embodiments, the LLDH is derived froman enzyme with native activity towards a substrate that is structurallysimilar to pyruvate.

Once a screen and/or selection is established and target genes (i.e.,for LLDH according to embodiments of the present disclosure) areidentified and integrated into the genome of recombinant host cells,recombinant host cells enter the directed evolution cycle, wherein thedirected evolution cycle comprises: (1) mutagenesis in response toselective pressure; (2) analysis of recombinant host cells in thegenerated library for measurable phenotypic differences that arise dueto selective pressure; and (3) isolation and characterization of evolvedvariants.

In some embodiments, acquisition of a mutation in the target geneenables the recombinant host cell to overcome the selective pressure. Insome embodiments, recombinant host cells are passaged throughout thecourse of mutagenesis with selective pressure. In various embodiments,the selective pressure comprises nutrient limitation, cellular toxicity,and extreme culture conditions that further comprise high or low pH,high or low temperature, and high or low osmotic pressure. In someembodiments, the recombinant host cells are initially propagated withoutselective pressure prior to mutagenesis.

After exposure to selective pressure for some period of time, theevolved or evolving strains are screened for a change in phenotype inresponse to selective pressure. Non-limiting examples of phenotypicchange include faster glucose consumption, faster cell growth, higherflux through a metabolic pathway or pathways, improved productyield/titer/productivity, decreased byproduct yield/titer, increasedtolerance to toxicity, or increased tolerance to extreme cultureconditions.

Enzyme Characterization

Protein variants that result from strain library generation andscreening are integrated into the genome of recombinant host cells andresulting strain variants are analyzed for LLDH activity. In someembodiments, iterative rounds of protein engineering are performed toproduce enzyme variants with optimized properties, wherein the iterativerounds of protein engineering comprise rational mutagenesis, randommutagenesis, and directed evolution. In these embodiments, selectvariants from preceding rounds of protein engineering are identified forfurther protein engineering. Non-limiting examples of such propertiescomprise improved enzyme kinetics for specificity and/or turnover,improved pathway flux, increased metabolite yield, decreased byproductyield. In some embodiments, culture medium or fermentation broth isanalyzed for the presence of metabolites such as L-lactic acid and/orbyproducts, wherein the method of analysis is HPLC (high-performanceliquid chromatography).

Ancillary Proteins

In addition to the L-lactic acid pathway enzymes, ancillary proteins areother proteins that are overexpressed in recombinant host cells of thepresent disclosure whose overexpression results in an increase inL-lactic acid as compared to control, or host cells that do notoverexpress said proteins. Ancillary proteins function outside theL-lactic acid pathway, wherein each ancillary protein plays a role thatindirectly boosts the recombinant host cell's ability to produceL-lactic acid. Ancillary proteins comprise any protein (excludingL-lactic acid pathway enzymes) of any structure or function that canincrease L-lactic acid yields, titers, or productivities whenoverexpressed. Non-limiting examples of classes of proteins includetranscription factors, transporters, scaffold proteins, proteins thatdecrease byproduct accumulation, and proteins that regenerate orsynthesize redox cofactors.

Provided herein in certain embodiments are recombinant host cellscomprising one or more heterologous nucleic acids encoding one or moreancillary proteins wherein said recombinant host cell is capable ofproducing higher L-lactic acid yields, titers, or productivities ascompared to control cells, or host cells that do not comprise saidheterologous nucleic acid(s). In some embodiments, that host recombinantcell naturally produces L-lactic acid, and in these cases, the L-lacticacid yields, titers, and/or productivities are increased. In otherembodiments, the recombinant host cell does not naturally produceL-lactic acid and thus comprises one or more heterologous nucleic acidsencoding one or more L-lactic acid pathway enzymes.

In certain embodiments of the present disclosure, the recombinant hostcells comprise one or more heterologous nucleic acids encoding one ormore L-lactic acid pathway enzymes and one or more heterologous nucleicacids encoding one or more ancillary proteins. In certain of theseembodiments, the recombinant host cells may be engineered to expressmore of these ancillary proteins. In these particular embodiments, theancillary proteins are expressed at a higher level (i.e., produced at ahigher amount as compared to cells that do not express said ancillaryproteins) and may be operatively linked to one or more exogenouspromoters or other regulatory elements.

In certain embodiments, recombinant host cells comprise both endogenousand heterologous nucleic acids encoding one or more L-lactic acidpathway enzymes and one or more ancillary proteins. In certainembodiments, the recombinant host cells comprise one or moreheterologous nucleic acids encoding one or more L-lactic acid pathwayenzymes and/or one or more ancillary proteins, and one or moreendogenous nucleic acids encoding one or more L-lactic acid pathwayenzymes and/or one or more ancillary proteins.

In certain embodiments, endogenous nucleic acids of ancillary proteinsare modified in situ (i.e., on chromosome in the host cell genome) toalter levels of expression, activity, or specificity. In someembodiments, heterologous nucleic acids are inserted into endogenousnucleic acids of ancillary proteins.

Ancillary proteins comprise proteins that recycle the redox cofactorsthat are produced during L-lactic acid pathway activity. Redox balanceis fundamental to sustained metabolism and cellular growth in livingorganisms. Intracellular redox potential is determined by redoxcofactors that facilitate the transfer of electrons from one molecule toanother within a cell. Redox cofactors in yeast comprise thenicotinamide adenine dinucleotides, NAD and NADP, the flavinnucleotides, FAD and FMN, and iron sulfur clusters (Fe-S clusters).

Redox constraints play a role in end-product formation. Additionalreducing power will typically be provided to produce compounds whosedegree of reduction is higher than that of the substrate. Conversely,producing compounds with a degree of reduction lower than that of thesubstrate will force the synthesis of byproducts with higher degrees ofreduction to compensate for excess reducing power generated fromsubstrate oxidation. Thus, maintaining redox neutrality may ensure highend-product yields. For example, the L-lactic acid pathway is redoxbalanced and there is no net formation of NAD(P)+ or NAD(P)H for eachmol of glucose converted to L-lactic acid in the cytosol.

The NAD and NADP cofactors are involved in electron transfer andcontribute to about 12% of all biochemical reactions in a cell (OstermanA., EcoSal Plus, 2009). NAD is assembled from L-aspartate,dihydroxyacetone phosphate (DHAP; glycerone), phosphoribosylpyrophosphate (PRPP) and ATP. The NADP is assembled in the same mannerand further phosphorylated. In some embodiments, recombinant host cellscomprise heterologous and/or endogenous nucleic acids encoding one ormore ancillary proteins facilitate NAD and NADP cofactor assembly. Insome embodiments, the ancillary proteins comprise one, more or allproteins suitable for use in accordance with methods of the presentdisclosure having NAD and/or NADP assembly capability, NAD and/or NADPtransfer capability, NAD and/or NADP chaperone capability, or anycombination thereof.

Similarly, Fe—S clusters facilitate various enzyme activities involvedwith electron transfer. Because both iron and sulfur atoms are highlyreactive and toxic to cells, Fe—S cluster assembly uses carefullycoordinated synthetic pathways in living cells. The three known pathwaysare the Isc (iron sulfur cluster) system, the Suf (sulfur formation)system, and the Nif (nitrogen fixation) system. Each of these systemshas a physiological role, yet several functional components are sharedbetween them. First, a cysteine desulfurase enzyme liberates sulfuratoms from free cysteine. Then, a scaffold protein receives theliberated sulfur for Fe—S cluster assembly. Finally, the Fe—S cluster istransferred to a target apoprotein. In some embodiments of the presentdisclosure, recombinant host cells comprise heterologous and/orendogenous nucleic acids encoding one or more ancillary proteins thatfacilitate Fe—S cluster assembly. In some embodiments, the ancillaryproteins comprise one, more or all proteins of the Isc system, the Sufsystem, the Nif system, or any combination thereof. In some embodiments,recombinant host cells comprise one or more heterologous nucleic acidsencoding one or more proteins suitable for use in accordance withmethods of the present disclosure having cysteine desulfurase activity,Fe—S cluster assembly capability, Fe—S cluster transfer capability, ironchaperone capability, or any combination thereof.

Another class of ancillary proteins useful for increasing L-lactic acidyields, titers, and/or productivities are organic acid transporterproteins. In some embodiments, recombinant host cells comprise one ormore heterologous and/or endogenous nucleic acids encoding one or moreorganic acid transporter proteins. In many embodiments, the organic acidtransporter is derived from a fungal source. In some embodiments, theorganic acid transporter is selected from the group comprisingSaccharomyces cerevisiae PDR12 (abbv. ScPDR12; UniProt ID: Q02785; SEQID NO: 2), Saccharomyces cerevisiae WAR1 (abbv. ScWAR1; UniProt ID:Q03631; SEQ ID NO: 3), Schizosaccharomyces pombe MAE1 (abbv. SpMAE1;UniProt ID; P50537; SEQ ID NO: 4), and Kluyveromyces marxianus PDC12(abbv. KmPDC12; UniProt ID: W0T9C6; SEQ ID NO: 5). In some embodiments,recombinant host cells comprise one or more heterologous nucleic acidsencoding one or more proteins suitable for use in accordance withmethods of the present disclosure have L-lactic acid transporteractivity. In some embodiments, recombinant host cells comprise one ormore heterologous nucleic acids encoding one or more proteins thatcomprise an amino acid sequence with at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%sequence identity with ScPDR12 (SEQ ID NO: 2), ScWAR1 (SEQ ID NO: 3),SpMAE1 (SEQ ID NO: 4), or KmPDC12 (SEQ ID NO: 5).

Decreasing or Eliminating Expression of Byproduct Pathway Enzymes

In an additional aspect of this disclosure, nucleic acids encodingbyproduct pathway enzymes can be disrupted in recombinant host cells ofthe present disclosure to increase L-lactic acid yields, productivities,and/or titers; and/or to decrease byproduct titers and/or yields ascompared to control cells, or host cells that express native/undisruptedlevels of said byproduct pathway enzymes. Byproduct pathway enzymescomprise any protein (excluding L-lactic acid pathway enzymes) of anystructure or function that can increase L-lactic acid product yields,titers, and/or productivities when disrupted because they utilizeintermediates or products of the L-lactic acid pathway. In addition,byproduct pathway enzymes also comprise any protein (excluding L-lacticacid pathway enzymes) of any structure or function that can decreaseundesired byproduct yields, titers, and/or productivities when disruptedbecause they utilize intermediates or products of the L-lactic acidpathway.

Byproducts that accumulate during L-lactic acid production can lead to:(1) lower L-lactic acid titers, productivities, and/or yields; and/or(2) accumulation of byproducts in the fermentation broth that increasethe difficulty of downstream purification processes. In someembodiments, recombinant host cells may comprise genetic disruptionsthat encompass alterations, deletions, knockouts, substitutions,promoter modifications, premature stop codons, or knock-downs thatdecrease byproduct accumulation. In some embodiments, recombinant hostcells comprising a disruption of one or more genes encoding a byproductpathway enzyme will have altered performance characteristics as comparedto cells without said genetic disruption(s), such as decreased oreliminated byproduct pathway enzyme expression, decreased or eliminatedbyproduct accumulation, improved L-lactic acid activity, alteredmetabolite flux through the L-lactic acid pathway, higher L-lactic acidtiters, productivities, yields, and/or altered cellular fitness.Decreasing byproduct formation can increase L-lactic acid activity,resulting in an increased amount of L-lactic acid produced. In manyembodiments, recombinant host cells of the present disclosure comprisingone or more genetic disruptions of one or more genes encoding abyproduct pathway enzyme produce an increased L-lactic acid titer ascompared to host cells that do not comprise said genetic disruption(s).In some of these embodiments, the L-lactic acid titer in thefermentation broth is increased by 0.5 g/L, 1 g/L, 2.5 g/L, 5 g/L, 7.5g/L, 10 g/L, or more than 10 g/L.

In addition to increasing L-lactic acid titers, decreasing byproductformation can also help increase L-lactic acid yields. Because yield isindependent of the volume of the fermentation broth, which can changeduring the course of a fermentation, it is often advantageous to measureL-lactic acid yields. In many embodiments, recombinant host cells of thepresent disclosure comprising one or more genetic disruptions of one ormore genes encoding byproduct pathway enzymes produce an increasedL-lactic acid yield as compared to host cells that do not comprise saidgenetic disruption. In some of these embodiments, the L-lactic acidyield is increased by 0.5%, 1%, 2.5%, 5%, 7.5%, 10%, or more than 10% (gL-lactic acid/g substrate). The substrate in this yield calculation isthe fermentation substrate, which is typically glucose, but may also beother, non-glucose substrates (for example, sucrose, glycerol, orpyruvate).

Increasing L-lactic acid can decrease manufacturing costs and canfurther work to disrupt genes encoding byproduct pathway enzymes inorder to decrease byproduct formation. Byproducts are typically unwantedchemicals, are disposed of as waste, and their disposal can involveelaborate processing steps and containment requirements. Therefore,decreasing byproduct formation can also lower production costs. In manyembodiments, recombinant host cells of the present disclosure comprisingone or more genetic disruptions of one or more genes encoding abyproduct pathway enzyme produces a lower byproduct titer as compared tohost cells that do not comprise said genetic disruption. In some ofthese embodiments, a recombinant host cell of the disclosure comprisinggenetic disruption of one or more byproduct pathway enzymes produces abyproduct titer that is 0.5 g/L, 1 g/L, 2.5 g/L, 5 g/L, 7.5 g/L, 10 g/L,or greater than 10 g/L less than host cells that do not comprise saidgenetic disruption.

In many embodiments, recombinant host cells of the present disclosurecomprising one or more genetic disruptions of one or more genes encodinga byproduct pathway enzyme produces a lower byproduct yield as comparedto host cells that do not comprise said genetic disruption(s). In someof these embodiments, recombinant host cells comprise genetic disruptionof one or more genes encoding byproduct pathway enzymes produce abyproduct yield that is 0.5%, 1%, 2.5%, 5%, 7.5%, 10%, or greater than10% (g byproduct/g substrate) less than host cells that do not comprisesaid genetic disruption. As with the L-lactic acid yield calculation,the substrate used in the byproduct yield calculation is the carbonsource provided to the fermentation; this is typically glucose, sucrose,or glycerol, but may be any carbon substrate.

Non-limiting examples of byproducts that arise due to consumption of anL-lactic acid pathway or a downstream pathway substrate, intermediate orproduct include acetaldehyde, acetyl-CoA and oxaloacetate. In the eventof a redox imbalance, an undesirable excess of reduced or oxidizedcofactors may also accumulate; thus, the redox cofactors NADH, NAD+,NADPH and NADP+ can also be considered byproducts.

A non-limiting list of enzyme-catalyzed reactions that utilize theL-lactic acid pathway substrate (i.e., pyruvate) are found in Table 1.Decreasing or eliminating expression of one, some or all of the genesencoding the enzymes in Table 1 can increase L-lactic acid productionand/or decrease byproduct production. In many cases, the product of theenzyme-catalyzed reactions provided in Table 1 can accumulate in thefermentation broth; in such cases, this indicates that expression of thenative gene encoding the listed enzyme should be reduced or eliminated.For example, the occurrence of acetaldehyde in the fermentation brothindicates that expression of a native gene encoding pyruvatedecarboxylase should be decreased or eliminated. In some cases, theproduct of the specific reaction listed in Table 1 is further converted,either spontaneously or through the action of other enzymes, into abyproduct that accumulates in the fermentation broth. In cases wherebyproduct accumulation is due to the activity of multiple enzymes, oneor more of the genes encoding the one or more byproduct pathway enzymescan be deleted or disrupted to reduce byproduct formation.

In some embodiments of the present disclosure, recombinant host cellscomprise microbial strains with decreased or eliminated expression ofone, some or all of the genes encoding enzymes listed in Table 1. Insome embodiments, recombinant host cells comprise microbial strains withdecreased byproduct accumulation wherein the byproducts are formedthrough the activity of one, some or all of the enzymes listed inTable 1. In some embodiments, recombinant host cells comprise microbialstrains with decreased expression of pyruvate-utilizing enzymes. In someembodiments, recombinant host cells comprise microbial strains withdecreased expression of L-lactic acid-utilizing enzymes. In someembodiments, recombinant host cells comprise microbial strains withinability to catabolize or breakdown L-lactic acid and/or L-lactic acid.

TABLE 1 Enzyme-catalyzed reactions that consume a substrate,intermediate or product of glycolysis or the L-lactic acid pathway.Substrate EC # Enzyme name Reaction Pyruvate 4.1.1.1 Pyruvate Pyruvate +H⁺ = decarboxylase Acetaldehyde + CO₂ Pyruvate n/a Pyruvate Pyruvate +CoA + Oxidized dehydrogenase cofactor = Acetyl-CoA + complex CO₂ +Reduced cofactor Pyruvate 6.4.1.1 Pyruvate Pyruvate + HCO₃ ⁻ +carboxylase ATP = Oxaloacetate + ADP + Phosphate + H⁺

Decreasing or Eliminating Expression of Certain Enzymes

Pyruvate decarboxylase catalyzes the irreversible/unidirectionalconversion of one molecule of pyruvate to one molecule of acetaldehydeand one molecule of CO₂. Pyruvate decarboxylase activity can lead to theformation of at least three undesirable pyruvate decarboxylase-basedbyproducts: acetaldehyde, acetate, and ethanol. There are at least 3pyruvate decarboxylase homologs in P. kudriavzevii: PkPDC1 (SEQ ID NO:6), PkPDC5 (SEQ ID NO: 7) and PkPDC6 (SEQ ID NO: 8); decreasing oreliminating expression of one or more of these homologs is useful forincreasing L-lactic acid production and/or decreasing accumulation ofpyruvate decarboxylase-based byproducts. As described above, homologousproteins share substantial sequence identity with each other. Anyprotein that is homologous to one, more, or all of the pyruvatedecarboxylases of the present disclosure (SEQ ID NOs: 6, 7 and 8) willshare substantial sequence identity one or more of these proteins.

In some embodiments, recombinant host cells comprise genetic disruptionsin one or more pyruvate decarboxylase homologs. As defined above,genetic disruptions encompass nucleic acid deletions, nucleic acidinsertions, nucleic acid substitutions, nucleic acid mutations,premature stop codons and promoter modifications. In some embodiments,recombinant host cells of the present disclosure comprise a geneticdisruption of a homologous pyruvate decarboxylase gene with at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or more than 95% homology when compared to PkPDC1(SEQ ID NO: 6), PkPDC5 (SEQ ID NO: 7) or PkPDC6 (SEQ ID NO: 8). In someof these embodiments, the recombinant host cell is a P. kudriavzeviistrain. In some embodiments, recombinant host cells comprise one or moregene disruptions that produce altered, decreased or eliminated activityin one, two or all three, pyruvate decarboxylase proteins. In some ofthese other embodiments, the recombinant host cell is a P. kudriavzeviistrain.

In some embodiments, recombinant host cells comprise heterologousnucleic acids encoding L-lactic pathway enzymes, and further compriseone or more genetic disruptions of one, more, or all of the pyruvatedecarboxylase homologs. In certain embodiments, acetaldehyde byproducttiter (i.e., g of byproduct/liter of fermentation volume) at the end offermentation is 10 g/L or less, 5 g/L or less, or 2.5 g/L or less. Incertain embodiments, acetaldehyde byproduct yield (i.e., percentage of gof byproduct/g of substrate) at the end of fermentation is 10% or less,5% or less, 2.5% or less, or 1% or less. In certain embodiments, acetatebyproduct titer at the end of fermentation is 10 g/L or less, 5 g/L orless, or 2.5 g/L or less. In certain embodiments, acetate byproductyield at the end of fermentation is 10% or less, 5% or less, 2.5% orless, or 1% or less. In some embodiments, ethanol byproduct titer at theend of a fermentation is 10 g/L or less, 5 g/L or less, or 2.5 g/L orless. In certain embodiments, ethanol byproduct yield at the end offermentation is 10% or less, 5% or less, 2.5% or less, or 1% or less.

The pyruvate dehydrogenase complex catalyzes the conversion of onemolecule of pyruvate, one molecule of coenzyme A and one molecule ofNAD+ to one molecule of acetyl-CoA, one molecule of CO₂ and one moleculeof NADH; in wild type P. kudriavzevii, this enzyme is localized in themitochondria. In most native microbes, the pyruvate dehydrogenasecomplex is used for aerobic metabolism of pyruvate to CO₂ through theactivity of the tricarboxylic acid cycle enzymes. Genetic disruption ofone or more genes encoding a protein subunit of the pyruvatedehydrogenase complex can decrease pyruvate dehydrogenase complexprotein activity or expression, consequently increasing L-lactic acidproduction and/or decreasing CO₂ byproduct formation. In someembodiments of the present disclosure, recombinant host cells comprisedecreased or eliminated expression and/or activity of one or morepyruvate dehydrogenase complex proteins. In some of these embodiments,recombinant host cells comprise decreased or eliminated expressionand/or activity of the E1 a-subunit of the pyruvate dehydrogenasecomplex (abbv. PkPDA1; SEQ ID NO: 9). In some embodiments, recombinanthost cells comprise one or more heterologous nucleic acids encoding oneor more proteins that comprise an amino acid sequence with at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, orat least 95% sequence identity with SEQ ID NO: 9. In some embodiments,the recombinant host cell is a P. kudriavzevii strain.

In many embodiments, wherein recombinant host cells comprise an L-lacticacid pathway and genetic disruption(s) that decrease or eliminateexpression and/or activity of one or more pyruvate dehydrogenase complexproteins, the L-lactic acid titer and/or yield is higher as compared torecombinant host cells that do not comprise said genetic disruption(s).In some of these embodiments, said recombinant host cells produce lesscarbon dioxide as compared to recombinant host cells that do notcomprise said genetic disruption(s). In some of these embodiments, therecombinant host cell's carbon dioxide yield (i.e., g-carbondioxide/g-glucose consumed) is lower as compared to recombinant hostcells that do not comprise said genetic disruption(s).

Additional byproducts can arise from intermediates in glycolysis.Glycerol is a common byproduct that occurs under conditions of excessNADH. NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8)catalyzes the conversion of one molecule of dihydroxyacetone phosphate(DHAP; glycerone phosphate) and one molecule of NAD(P)H to one moleculeof glycerol 3-phosphate and one molecule of NAD(P)+, leading to theformation of the undesired byproduct glycerol. In P. kudriavzevii,NAD-dependent glycerol-3-phosphate dehydrogenase activity is the Gpd1protein (abbv. PkGPD1; SEQ ID NO: 10).

Decreasing or eliminating the expression of PkGPD1 or its homologs canbe useful for decreasing glycerol byproduct accumulation. In someembodiments of the present disclosure, recombinant host cells compriseone or more genetic disruptions in one or more nucleic acids encoding aglycerol-3-phosphate dehydrogenase that gives rise to decreased, alteredor eliminated expression and/or protein activity. In embodiments wherethe recombinant host cell is a P. kudriavzevii strain, theglycerol-3-phosphate dehydrogenase is PkGPD1.

In some embodiments, recombinant host cells comprise heterologousnucleic acids encoding L-lactic acid pathway enzymes, and furthercomprise one or more genetic disruptions in PkGPD1 (SEQ ID NO: 10), orin one, more, or all PkGPD1 homologs wherein several amino acids in thePkGPD1 homologs are conserved. In certain embodiments, glycerolbyproduct titer at the end of fermentation is at a titer of 10 g/L orless, 5 g/L or less, or 2.5 g/L or less. In certain embodiments,glycerol byproduct yield at the end of fermentation is 10% or less, 5%or less, 2.5% or less, or 1% or less.

The LLDH of the L-lactic acid pathway catalyzes the conversion of onemolecule of pyruvate and one molecule of NAD(P)H to one molecule ofL-lactic acid and one molecule of NAD(P)+. In order to ensure NAD(P)Havailability does not become a limiting factor in this reaction, hostcell endogenous proteins with NAD(P)H dehydrogenase activity aredecreased or eliminated in some embodiments of the present disclosure.

NAD(P)H dehydrogenases belong to the enzyme family of oxidoreductasesthat work with other electron acceptors to catalyze the transfer ofelectrons from one molecule to another. NAD(P)H dehydrogenases typicallyfunction in the shuttling of electrons from the cytosol to the electrontransport chain where they are used to generate ATP and water from O₂.NADH dehydrogenases utilize the NADH cofactor while NADPH dehydrogenasesutilize the NADPH cofactor. NAD(P)H dehydrogenases typically havespecificity to either NADH or NADPH, although it is possible for someengineered as well as natural NAD(P)H dehydrogenases to be able to bindeither cofactor with varying affinities and utilize either cofactor withvarying catalytic efficiencies.

In accordance with the present disclosure, it may be desirable todecrease or eliminate host cell endogenous NAD(P)H dehydrogenaseexpression so that the L-lactic acid pathway is not limited by theavailability of NAD(P)H cofactor. Decreasing or eliminating expressionof one or more homologs of NAD(P)H dehydrogenase is useful forincreasing L-lactic acid production. In embodiments where the LLDHutilizes the NADH cofactor, the expression of more host cell endogenousNADH dehydrogenase enzymes is decreased or eliminated. In embodimentswhere the LLDH utilizes the NADPH cofactor, the expression of one ormore host cell endogenous NADPH dehydrogenase enzymes is decreased oreliminated. In some embodiments, the NAD(P)H dehydrogenase is themitochondrial external NADH dehydrogenase. In some embodiments, theNAD(P)H dehydrogenase is the P. kudriavzevii Nde1 protein (abbv. PkNDE1;SEQ ID NO: 11). In some embodiments, recombinant host cells comprise oneor more heterologous nucleic acids encoding one or more proteins thatcomprise an amino acid sequence with at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%sequence identity with SEQ ID NO: 11. In embodiments wherein therecombinant host cells comprise an L-lactic acid pathway and a decreaseor elimination of one or more copies of host cell endogenous NAD(P)Hdehydrogenase, the recombinant host cells further comprise increasedL-lactic acid titer, L-lactic acid yield, and/or L-lactic acidproductivity.

Genetic Engineering

Expression of L-lactic acid pathway enzymes is achieved by transforminghost cells with exogenous nucleic acids encoding L-lactic acid pathwayenzymes, producing recombinant host cells of the present disclosure. Thesame is true for expression of ancillary proteins. Any method can beused to introduce exogenous nucleic acids into a host cell to produce arecombinant host cell of the present disclosure. Many such methods areknown to practitioners in the art. Some examples compriseelectroporation, chemical transformation, and conjugation. Some examplescomprise electroporation, chemical transformation, and conjugation.After exogenous nucleic acids enter the host cell, nucleic acids mayintegrate in to the cell genome via homologous recombination.

Recombinant host cells of the present disclosure may comprise one ormore exogenous nucleic acid molecules/elements, as well as single ormultiple copies of a particular exogenous nucleic acid molecule/elementas described herein. These molecules/elements comprise transcriptionalpromoters, transcriptional terminators, protein coding regions, openreading frames, regulatory sites, flanking sequences for homologousrecombination, and intergenic sequences.

Exogenous nucleic acids can be maintained by recombinant host cells invarious ways. In some embodiments, exogenous nucleic acids areintegrated into the host cell genome. In other embodiments, exogenousnucleic acids are maintained in an episomal state that can bepropagated, either stably or transiently, to daughter cells. Exogenousnucleic acids may comprise selectable markers to ensure propagation. Insome embodiments, the exogenous nucleic acids are maintained inrecombinant host cells with selectable markers. In some embodiments, theselectable markers are removed and exogenous nucleic acids aremaintained in a recombinant host cell strain without selection. In someembodiments, removal of selectable markers is advantageous fordownstream processing and purification of the fermentation product.

In some embodiments, endogenous nucleic acids (i.e., genomic orchromosomal elements of a host cell), are genetically disrupted toalter, mutate, modify, modulate, disrupt, enhance, remove, or inactivatea gene product. In some embodiments, genetic disruptions alterexpression or activity of proteins native to a host cell. In someembodiments, genetic disruptions circumvent unwanted byproduct formationor byproduct accumulation. Genetic disruptions occur according to theprinciple of homologous recombination via methods well known in the art.Disrupted endogenous nucleic acids can comprise open reading frames aswell as genetic material that is not translated into protein. In someembodiments, one or more marker genes replace deleted genes byhomologous recombination. In some of these embodiments, the one or moremarker genes are later removed from the chromosome using techniquesknown to practitioners in the art.

Producing L-Lactic Acid and L-Lactate Salts with Recombinant Host Cells

Methods are provided herein for producing L-lactic acid or L-lactatesalts from recombinant host cells of the present disclosure. In certainembodiments, the methods comprise the steps of: (1) culturingrecombinant host cells as provided by the present disclosure in afermentation broth containing at least one carbon source and onenitrogen source under conditions such that L-lactic acid or L-lactate isproduced; and (2) recovering the L-lactate, L-lactic acid or L-lactatesalt from the fermentation broth. In some embodiments, the L-lactic acidis first converted to an L-lactate salt before the L-lactate salt isrecovered from the fermentation broth. In some embodiments, theL-lactate acid or L-lactate salt is first converted to a downstreamproduct before the downstream product is recovered from the fermentationbroth.

Any of the recombinant host cells of the present disclosure can becultured to produce and/or secrete L-lactate (i.e., L-lactic acid andL-lactate salt). As disclosed herein, the L-lactate can then beesterified and distilled to generate a purified ester.

Materials and methods for the maintenance and growth of microbes, aswell as fermentation conditions, are well known to practitioners ofordinary skill in the art. It is understood that consideration willtypically be given to appropriate culture medium, pH, temperature,revival of frozen stocks, growth of seed cultures and seed trains, andrequirements for aerobic, microaerobic, or anaerobic conditions,depending on the specific requirements of the host cells, thefermentation, and process flows.

The methods of producing L-lactate provided herein may be performed in asuitable fermentation broth in a suitable bioreactor such as afermentation vessel, including but not limited to a culture plate, aflask, or a fermenter. Further, the methods can be performed at anyscale of fermentation known to support microbial production ofsmall-molecules on an industrial scale. Any suitable fermenter may beused including a stirred tank fermenter, an airlift fermenter, a bubblecolumn fermenter, a fixed bed bioreactor, or any combination thereof.

In some embodiments of the present disclosure, the fermentation broth isany fermentation broth in which a recombinant host cell capable ofproducing L-lactate according to the present disclosure, and can subsist(i.e., maintain growth, viability, and/or catabolize glucose or othercarbon source). In some embodiments, the fermentation broth is anaqueous medium comprising assimilable carbon, nitrogen, and phosphatesources. Such a medium can also comprise appropriate salts, minerals,metals, and other nutrients. In some embodiments, the carbon source andeach of the essential cell nutrients are provided to the fermentationbroth incrementally or continuously, and each essential cell nutrient ismaintained at essentially the minimum level for efficient assimilationby growing cells. For example, cell growth procedures comprise batchfermentation, fed-batch fermentation with batch separation, fed-batchfermentation with continuous separation, and continuous fermentationwith continuous separation. These procedures are well known topractitioners of ordinary skill in the art.

In some embodiments of the present disclosure, the handling andculturing of recombinant host cells to produce L-lactate may be dividedup into phases, such as growth phase, production phase, and/or recoveryphase. The following paragraphs provide examples of features or purposesthat may relate to these different phases. These features or purposesmay vary based on the recombinant host cells used, the desired L-lactateyield, titer, and/or productivity, or other factors. While it may bebeneficial in some embodiments for the L-lactic acid pathway enzymes,ancillary proteins and/or endogenous host cell proteins to beconstitutively expressed, other embodiments may comprise selectiveexpression or repression of any or all of the aforementioned proteins.

During growth phase, recombinant host cells may be cultured to focus ongrowing cell biomass by utilizing the carbon source provided. In someembodiments, expression of L-lactic acid pathway enzymes and/orancillary proteins are repressed or uninduced. In some embodiments, noappreciable amount of L-lactate is made. In some embodiments, proteinsthat contribute to cell growth and/or cellular processes may beselectively expressed.

During production phase, however, recombinant host cells may be culturedto stop producing cell biomass and to focus on L-lactate biosynthesis byutilizing the carbon source provided. In some embodiments, L-lactic acidpathway enzymes, and/or ancillary proteins may be selectively expressedduring production to generate high product titers, yields andproductivities. The production phase is synonymous with fermentation,fermentation run or fermentation phase.

In some embodiments, the growth and production phases take place at thesame time. In other embodiments, the growth and production phases areseparate. While in some embodiments, product is made exclusively duringproduction phase, in other embodiments some product is made duringgrowth phase before production phase begins.

The recovery phase marks the end of the production phase, during whichcellular biomass is separated from fermentation broth and L-lactate ispurified from fermentation broth. In some fermentation process, forexample, fill-draw and continuous fermentations, there may be multiplerecovery phases where fermentation broth containing biomass and L-lacticacid are removed from the fermentation system. The draws of fermentationbroth may be processed independently or may be stored, pooled, andprocessed together. In other fermentation processes, for example, batchand fed-batch fermentations, there may only be a single recovery phase.

Fermentation procedures are particularly useful for the biosyntheticproduction of commercial L-lactate. Fermentation procedures can bescaled up for manufacturing L-lactate and fermentation procedurescomprise, for example, fed-batch fermentation and batch productseparation; fed-batch fermentation and continuous product separation;batch fermentation and batch product separation; and continuousfermentation and continuous product separation.

The carbon source provided to the fermentation can be any carbon sourcethat can be fermented by recombinant host cells. Suitable carbon sourcesinclude, but are not limited to, monosaccharides, disaccharides,polysaccharides, glycerol, acetate, ethanol, methanol, methane, or oneor more combinations thereof. Monosaccharides suitable for use inaccordance to the methods of the present disclosure include, but are notlimited to, dextrose (glucose), fructose, galactose, xylose, arabinose,and any combination thereof. Disaccharides suitable for use inaccordance to the methods of the present disclosure include, but are notlimited to, sucrose, lactose, maltose, trehalose, cellobiose, and anycombination thereof. Polysaccharides suitable for use in accordance tothe methods of the present disclosure include, but are not limited to,starch, glycogen, cellulose, and combinations thereof. In someembodiments, the carbon source is dextrose. In other embodiments, thecarbon source is sucrose. In some embodiments, mixtures of some or allthe aforementioned carbon sources can be used in fermentation.

The pH of the fermentation broth can be controlled by the addition ofacid or base to the culture medium. In some embodiments, fermentation pHis controlled at the beginning of fermentation and then allowed to dropas L-lactic acid accumulates in the broth, minimizing the amount of baseadded to the fermentation (thereby improving process economics) as wellas minimizing the amount of salt formed. Specifically, the pH duringfermentation is maintained in the range of 2-8. At the end offermentation, the final pH is in the range of 2-5. Non-limiting examplesof suitable acids used to control fermentation pH include aspartic acid,acetic acid, hydrochloric acid, and sulfuric acid. Non-limiting examplesof suitable bases used to control fermentation pH include sodiumbicarbonate (NaHCO₃), sodium hydroxide (NaOH), potassium bicarbonate(KHCO₃), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)₂), calciumcarbonate (CaCO₃), ammonia, ammonium hydroxide, and diammoniumphosphate. In some embodiments, a concentrated acid or concentrated baseis used to limit dilution of the fermentation broth.

Base cations and L-lactate anions react to form ionic compounds infermentation broths. For example, base Na⁺ cations and L-lactate anionsreact to form sodium L-lactate. In some embodiments, the ionic compoundsformed by base cations and L-lactate anions are soluble in fermentationbroth. In other embodiments, the ionic compounds formed by base cationsand L-lactate anions are insoluble salts and may crystallize in thefermentation broth.

The temperature of the fermentation broth can be any temperaturesuitable for growth of the recombinant host cells and/or production ofL-lactic acid. In some embodiments, during L-lactic acid production, thefermentation broth is maintained within a temperature range of fromabout 20° C. to about 45° C., or in the range of from about 30° C. toabout 42° C.

The present disclosure provides methods to achieve high L-lactic acidyields, titers, and productivities under aerobic conditions. Typically,lactic acid can be efficiently produced under anaerobic or microaerobicfermentation conditions. Under aerobic conditions, microbes willcommonly use molecular oxygen as an electron acceptor to reoxidizeNAD(P)H cofactors in the electron transport chain of the mitochondria,generating ATP useful for growth and maintenance of cellular functions.In the absence of oxygen, microbes can only reoxidize the NAD(P)Hresulting from glycolysis through the activity of product pathways thatare redox balanced, one of which is production of L-lactic acid fromglucose. There are several downsides to anaerobic (or oxygen limited)production of lactic acid. First, glucose consumption rates are lower,leading to lower fermentation productivities. Second, insufficient ATPis generated to concomitantly maintain cellular activities, exportlactic acid, support high lactic acid titers, and support pathwayactivity under low pH fermentation conditions. Thus, it would beadvantageous to produce lactic acid under aerobic conditions where theATP generated from aerobic respiration can be used to increasefermentation metrics. For example, aerobically generated ATP can be usedby the cell to tolerate higher L-lactic acid titers and lowerfermentation pH ranges, which translate to achieving higher L-lacticacid yields, titers, and/or productivities as compared to anaerobic oroxygen-limited fermentations.

As described previously, the L-lactic acid pathway is redox balanced(i.e., conversion of glucose to L-lactic acid results in no netNAD(P)H). Thus, in recombinant host cells comprising deletion ordisruption of the external NAD(P)H dehydrogenase responsible for passingelectrons from cytosolic NAD(P)H into the electron transport chain, andexpression of a LLDH in the cytosol, the primary route for the cell toreoxidize cytosolic NAD(P)H is through lactic acid production.Additional genetic modifications can be introduced into recombinant hostcells to modulate the flux of TCA cycle substrates (typically pyruvate)into the mitochondria, where the TCA cycle substrates are aerobicallyrespired to carbon dioxide along with concomitant generation of NADH(and potentially other redox cofactors) through the activity of theelectron transport chain. Thus, by controlling the flux of TCA cyclesubstrates into the mitochondria, the amount of glucose aerobicallyrespired to carbon dioxide can be controlled such that sufficient ATP isgenerated to support high lactic acid titers and/or productivitieswithout detracting from lactic acid yields from glucose.

During cultivation, aeration and agitation conditions are selected toproduce an oxygen transfer rate (OTR; rate of dissolution of dissolvedoxygen in a fermentation medium) that results in high L-lactic acidtiters at low final fermentation pH values. In various embodiments,fermentation conditions are selected to produce an OTR of greater than 5mmol/L/hr. In some embodiments, fermentation conditions are selected toproduce an OTR of greater than 10 mmol/L/hr, 20 mmol/L/hr, 30 mmol/L/hr,40 mmol/L/hr, 50 mmol/L/hr, 75 mmol/L/hr, 100 mmol/L/hr, 125 mmol/L/hr,150 mmol/L/hr, 175 mmol/L/hr, or 200 mmol/L/hr. OTR as used hereinrefers to the volumetric rate at which oxygen is consumed during thefermentation. Inlet and outlet oxygen concentrations can be measured byexhaust gas analysis, for example by mass spectrometers. OTR can becalculated using the Direct Method described in Bioreaction EngineeringPrinciples 3rd Edition, 2011, Spring Science+Business Media, p. 449. Therecombinant host cells of the present disclosure are able to produceL-lactic acid under a wide range of oxygen concentrations.

A high yield of L-lactate from the provided carbon source(s) isdesirable to decrease the production cost. As used herein, yield iscalculated as the percentage of the mass of carbon source catabolized byrecombinant host cells of the present disclosure and used to produceL-lactate. In some cases, only a fraction of the carbon source providedto a fermentation is catabolized by the cells, and the remainder isfound unconsumed in the fermentation broth or is consumed bycontaminating microbes in the fermentation. Thus, it is important toensure that fermentation is both substantially pure of contaminatingmicrobes and that the concentration of unconsumed carbon source at thecompletion of the fermentation is measured. For example, if 100 grams ofglucose is fed into the fermentation, and at the end of the fermentation25 grams of L-lactic acid are produced and there remains 10 grams ofglucose, the L-lactate yield is 27.7% (i.e., 25 grams L-lactate from 90grams glucose). In certain embodiments of the methods provided herein,the final yield of L-lactic acid on the carbon source is at least 10%,at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, or greater than 80%. In certainembodiments, the recombinant host cells provided herein are capable ofproducing at least 70%, at least 75%, or greater than 80% by weight ofcarbon source to L-lactate. When an L-lactate salt is found in thefermentation broth, the L-lactic acid yield can be determined bycalculating the mols of L-lactate salt present and adjusting for themolecular weight difference between the L-lactate salt and L-lacticacid.

In addition to yield, the titer (or concentration), of L-lactateproduced in the fermentation is another useful metric for production.Generally speaking, titer is provided as grams of product (for example,L-lactate) per liter of fermentation broth (i.e., g/L). In someembodiments, the lactic acid titer is at least 1 g/L, at least 5 g/L, atleast 10 g/L, at least 15 g/L, at least 20 g/L, at least 25 g/L, atleast 30 g/L, at least 40 g/L, at least 50 g/L, at least 60 g/L, atleast 70 g/L, at least 80 g/L, at least 90 g/L, at least 100 g/L, atleast 125 g/L, at least 150 g/L, or greater than 150 g/L at some pointduring the fermentation, or at the conclusion of the fermentation. Insome embodiments, the L-lactic acid titer at the conclusion of thefermentation is greater than 100 g/L. In some embodiments, the L-lacticacid titer at the conclusion of the fermentation is greater than 125g/L. In some embodiments, the L-lactic acid titer at the conclusion ofthe fermentation is greater than 150 g/L. As with yield calculations, aL-lactic acid titer can be calculated from the L-lactate salt titer byadjusting for molecular weight differences between the L-lactate saltand L-lactic acid.

Further, productivity, or the rate of product (i.e., L-lactate)formation, is useful for decreasing production cost. Generally speaking,productivity is provided as grams product produced per liter offermentation broth per hour (i.e., g/L/hr). In some embodiments,L-lactic acid productivity is at least 0.1 g/L/hr, at least 0.25 g/L/hr,at least 0.5 g/L/hr, at least 0.75 g/L/hr, at least 1.0 g/L/hr, at least1.25 g/L/hr, at least 1.25 g/L/hr, at least 1.5 g/L/hr, at least 2.0g/L/hr, at least 3.0 g/L/hr, at least 4.0 g/L/hr, at least 5.0 g/L/hr,at least 6.0 g/L/hr or greater than 6.0 g/L/hr over some time periodduring the fermentation. In some embodiments, the L-lactic acidproductivity is at least 3 g/L/hr. In some embodiments, the L-lacticacid productivity is at least 4 g/L/hr. In some embodiments, theL-lactic acid productivity is at least 5 g/L/hr.

HPLC is an appropriate method to determine the amount of L-lactateand/or produced, the amount of any byproducts produced (for example,organic acids and alcohols), the amount of any pathway metabolite orintermediate produced, and the amount of unconsumed glucose left in thefermentation broth. Aliquots of fermentation broth can be isolated foranalysis at any time during fermentation, as well as at the end offermentation. Briefly, molecules in the fermentation broth are firstseparated by liquid chromatography (LC); then, specific liquid fractionsare selected for analysis using an appropriate method of detection (forexample, UV-VIS, refractive index, and/or photodiode array detectors).In some embodiments of the present disclosure, an organic acid salt (forexample, L-lactate) is the fermentative product present in thefermentation broth. The salt is acidified before or during HPLC analysisto produce L-lactic acid. Hence, the acid concentration calculated byHPLC analysis can be used to calculate the salt titer in thefermentation broth by adjusting for difference in molecular weightbetween the two compounds.

Gas chromatography-mass spectrometry (GC-MS) is also an appropriatemethod to determine the amount of target product and byproducts,particularly if they are volatile. Samples of fermentation can beisolated any time during and after fermentation and volatile compoundsin the headspace can be extracted for analysis. Non-volatile compoundsin the fermentation medium (for example, organic acids) can also beanalyzed by GC-MS after derivatization (i.e., chemical alteration) fordetection by GC-MS. Non-volatile compounds can also be extracted fromfermentation medium by sufficiently increasing the temperature of thefermentation medium, causing non-volatile compounds to transition intogas phase for detection by GC-MS. Molecules are carried by an inert gascarries as they move through a column for separation and then arrive ata detector.

Purification of L-Lactic Acid and L-Lactate Salts

The present disclosure describes the methods for purifying and analyzingfermentation product synthesized by recombinant cells of the presentdisclosure, wherein the fermentation product comprises L-lactic acid andL-lactates. The methods comprise separating soluble fermentation productfrom fermentation broth, cells, cell debris and soluble impurities, andisolating the soluble fermentation product. The fermentation product isanalyzed for relative amounts of L- and L-lactic acid enantiomers. Insome examples, the methods may also comprise converting fermentationproduct from soluble form to insoluble, crystalline form, and isolatingthe crystalline fermentation product.

At the end of fermentation, the fermentation broth contains fermentationproduct, in soluble and/or insoluble forms, together with biomass andsoluble impurities that comprise salts, proteins, unconverted sugars,and other impurities including color bodies. Biomass and solubleimpurities are removed via a series of purification steps. In certainembodiments of the present disclosure, purification steps may comprisecentrifugation, microfiltration, ultrafiltration, nanofiltration,diafiltration, ion exchange, crystallization, and any combinationthereof. In some of these embodiments, ion exchange resins andnanofiltration membranes are used as polishing steps to remove traceamounts of soluble impurities, unconverted sugars and color bodies.

In some embodiments, the process of purifying fermentation product(i.e., L-lactic acid and L-lactates) comprises a step of separating aliquid fraction containing fermentation product from a solid fractionthat contains cells and cell debris. For this separation, any amount offermentation broth can be processed, including the entirety of thefermentation broth. The amount of fermentation broth processed candepend on the type of fermentation process used, such as batch orcontinuous fermentation processes. In various embodiments, removal ofcells and cell debris can be accomplished, for example, viacentrifugation using specific g-forces and residence times, and/orfiltration using molecular weight cutoffs that are suitable forefficiently separating the liquid fraction containing fermentationproduct from the solid fraction that contains cells and cell debris. Insome embodiments, removal of cells and cell debris is repeated at leastonce at one or in more than one step in the methods provided herein.

In some embodiments, centrifugation is used to provide a liquid fractioncomprising fermentation product that is substantially free of cells.Many types of centrifuges useful for the removal of cells and solidsfrom fermentation broth are known to those skilled in the art, includingdisc-stack and decanter centrifuges. Centrifuges are well suited forseparating solids with a particle size of between 0.5 mm to 500 mm anddensity greater than that of the liquid phase (ca. 1.0 g/mL). Yeastcells, as a non-limiting example of a fermentation product-producingmicrobe, typically have a particle size between 4-6 mm and a density ofaround 1.1 g/mL; therefore, centrifugation is well suited for removingyeast cells from fermentation broth.

In some embodiments, a disc-stack centrifuge is used to provide a liquidfraction comprising fermentation product that substantially free ofcells. A disc stack centrifuge separates solids, which are dischargedintermittently during operation, from liquids, typically in a continuousprocess. A disc-stack centrifuge is well suited for separating soft,non-abrasive solids, including cells. In some embodiments, a decantercentrifuge is used to provide a liquid fraction comprising fermentationproduct that is substantially free of cells. A decanter centrifuge cantypically process larger amounts of solids and is often used instead ofa disc-stack centrifuge for processing fermentation broth when the cellmass and other solids exceeds about 3% w/w.

Other methods can be used in addition to, or alone, with the abovecentrifugation processes. For example, microfiltration is also aneffective means to remove cells from fermentation broth. Microfiltrationcomprises filtering the fermentation broth through a membrane havingpore sizes from about 0.5 mm to about 5 mm. In some embodiments,microfiltration is used to provide a liquid fraction comprisingfermentation product that is substantially free of cells.

In some embodiments, cells removed by centrifugation and/ormicrofiltration are recycled back into the fermentation. Recycling cellsback into the fermentation can increase fermentation product yield sinceless carbon source (for example, glucose) will typically be used togenerate new cells. Additionally, recycling cells back into thefermentation can also increase fermentation product productivity sincethe concentration of cells producing L-lactic acid and/or L-lactate inthe fermenter can be increased.

While suitable for removing cells, centrifugation and microfiltrationare not generally effective at removing cells debris, proteins, DNA andother smaller molecular weight compounds from the fermentation broth.Ultrafiltration is a process similar to microfiltration, but themembrane has pore sizes ranging from about 0.005 mm to 0.1 mm. This poresize equates to a molecular weight cut-off (the size of macromoleculethat will be ca. 90% retained by the membrane) from about 1,000 Daltonsto about 200,000 Daltons. The ultrafiltration permeate will containlow-molecular weight compounds, including fermentation product andvarious other soluble salts while the ultrafiltration retentate willcontain the majority of residual cell debris, DNA, and proteins. In someembodiments, ultrafiltration is used to provide a liquid fractioncomprising L-lactate salts that is substantially free of cell debris andproteins.

In some embodiments, nanofiltration is used to separate out certaincontaminating salts, sugars, color forming bodies, and other organiccompounds present in clarified fermentation broth containingfermentation product (i.e., L-lactic acid and L-lactates). Innanofiltration, the clarified fermentation broth (i.e., the fermentationbroth resulting from the combination of centrifugation, microfiltration,and/or ultrafiltration steps described above) is filtered through amembrane having pore sizes ranging from 0.0005 mm to 0.005 mm, equatingto a molecular weight cut-off of about 100 Daltons to about 2,000Daltons. Nanofiltration can be useful for removing divalent andmultivalent ions, maltose and other disaccharides (for example,sucrose), polysaccharides, and other complex molecules with a molecularweight larger than fermentation product (for example, sodium L-lactate112.06 g/mol, magnesium L-lactate 202.45 g/mol, calcium L-lactate 218.22g/mol, potassium L-lactate 128.17 g/mol). Non-limiting examples ofnanofiltration materials include ceramic membranes, metal membranes,polymer membranes, and composite membranes.

In some embodiments, ion exchange is used to remove specificcontaminating salts present in clarified fermentation broth containingfermentation product. Ion exchange elements can take the form of resinbeads as well as membranes. Frequently, the resins are cast in the formof porous beads. The resins can be cross-linked polymers having activegroups in the form of electrically charged sites. At these sites, ionsof opposite charge are attracted but may be replaced by other ionsdepending on their relative concentrations and affinities for the sites.Ion exchangers can be cationic or anionic. Factors that determine theefficiency of a given ion exchange resin comprise the favorability for agiven ion, and the number of active sites available.

A combination of nanofiltration and ion exchange steps can be combinedto produce a purified solution of fermentation product from clarifiedfermentation broth.

The purified solution of fermentation product (i.e., L-lactic acid andL-lactates) as described thus far can be analyzed for enantiomericpurity. In some embodiments, the purified fermentation product isevaluated using a chiral gas chromatography method known topractitioners of ordinary skill in the art. Briefly, the acid and analcohol are added to the purified solution of fermentation to producederivatized products of lactic acid. The derivatized products of lacticacid are then prepared for analysis by gas chromatography (GC) using aflame ionization detector (FID). Separation of derivatized enantiomersis achieved using Agilent CycloSil-B chiral capillary column andstandard operating procedures, or other columns with equivalentcapabilities and their respective standard operating procedures.Enantiomeric purity is defined as100%×{(L-enantiomer)/(D-enantiomer+L-enantiomer)}.

Fermentation products (i.e., L-lactic acid and L-lactates) purified asdescribed thus far can be crystallized to further remove water and anyremaining trace, water-soluble impurities. The solution of purifiedfermentation product as produced by the aforementioned steps can be thenfed to the fermentation product crystallization step. In someembodiments of the present disclosure, it is desirable to recover themajority of the L-lactic acid in the insoluble, crystallized form with aminor fraction of L-lactic acid remaining in the mother liquor.

In some embodiments of the present disclosure, the temperature of themother liquor is changed to facilitate fermentation productcrystallization. In some embodiments, the mother liquor is cooled to atemperature below 20° C. to decrease fermentation product solubility. Insome these embodiments, the mother liquor is heated to evaporate excesswater. In some of these embodiments, evaporative crystallization isused, as it offers a high yield of fermentation product and prevents theformation of stable gels, which may occur if temperature is reducedbelow the gelling point of concentrated fermentation product solutions.In some of these embodiments, fermentation product crystallization isachieved by combining various heating and cooling steps. In some ofthese embodiments, supersaturation is achieved by evaporativecrystallization wherein the solute is more concentrated in a bulksolvent that is normally possible under given conditions of temperatureand pressure; increased supersaturation of fermentation product in themother liquor causes the fermentation product to crystallize.Non-limiting examples of crystallizers include forced circulationcrystallizers, turbulence/draft tube and baffle crystallizers, inducedcirculation crystallizers and Oslo-type crystallizers.

In some embodiments of the present disclosure, the aforementionedheating step, cooling step and change in pH are combined in various waysto crystallize fermentation product, and modified as needed, as apparentto practitioners skilled in the art.

Fermentation product crystals can be isolated from the mother liquor byany technique apparent to those of skill in the art. In some embodimentsof the present disclosure, fermentation product crystals are isolatedbased on size, weight, density, or combinations thereof. Fermentationproduct crystal isolation based on size can be accomplished, forexample, via filtration, using a filter with a specific particle sizecutoff. Fermentation product crystal isolation based on weight ordensity can be accomplished, for example, via gravitational settling orcentrifugation, using, for example, a settler, decanter centrifuge,disc-stack centrifuge, basket centrifuge, or hydrocyclone whereinsuitable g-forces and settling or centrifugation times can be determinedusing methods known in the art. In some embodiments, fermentationproduct crystals are isolated from the mother liquor via settling forfrom 30 minutes to 2 hours at a g-force of 1. In other embodiments,L-lactate salt crystals are isolated from the fermentation broth viacentrifugation for 20 seconds to 60 seconds at a g-force of from 275 x-gto 1,000 x-g.

Following isolation from the mother liquor, fermentation productcrystals are wet with residual mother liquor that coats the crystals.Thus, in some embodiments, it is useful to wash the fermentation productcrystals with water to remove these trace impurities that may be in themother liquor. When washing fermentation product crystals, it issometimes desired to minimize the dissolution of isolated crystals inthe wash water; for this reason, in some embodiments, cold wash (around4° C.) water is generally used. Additionally, it can be desired tominimize the amount of wash water used to minimize crystal dissolution.In many embodiments, less than 10% w/w wash water is used to wash thefermentation product crystals.

In some embodiments, the methods further comprise the step of removingimpurities from fermentation product crystals. Impurities may react withfermentation product crystals and reduce final yields or contribute tofermentation product crystals of lesser purity that limits industrialutility. Non-limiting examples of impurities include acetic acid,succinic acid, malic acid, ethanol, glycerol, citric acid, and propionicacid. In some embodiments, removal of such impurities is accomplished bydissolving the isolated fermentation product crystals into an aqueoussolution and recrystallizing the fermentation product. A non-limitingexample of dissolving and recrystallizing fermentation product crystalscan include dissolving the fermentation product in water and evaporatingthe resulting aqueous solution (as mentioned above), and finallyre-isolating the fermentation product crystals by filtration and/orcentrifugation. None, one, or more than one cycle of fermentationproduct recrystallization may be used so long as the resultingfermentation product are of suitable quality for subsequentesterification. In some embodiments, no fermentation productrecrystallizations are performed. In other embodiments, one fermentationproduct recrystallization is performed. In still further embodiments,more than one fermentation product recrystallization is performed.

In some embodiments of the present disclosure, fermentation productcrystals are dewatered using a combination of screening and dryingmethods apparent to practitioners skilled in the art. In some of theseembodiments, crystal dewatering steps comprise centrifugation, beltdrying, filtration, application of vacuum, or a combination thereof. Insome of these embodiments, vacuum is applied at 20 mm of Hg belowatmospheric pressure. Suitable devices for crystal dewatering maycomprise a Horizontal Vacuum Belt Filter (HVBF) or a Rotary Drum VacuumFilter (RDVF). Fermentation product crystal isolation based on size canbe accomplished, for example, via filtration, using, for example, afilter press, candlestick filter, or other industrially used filtrationsystem with appropriate molecular weight cutoff. Fermentation productcrystal isolation based on weight or density can be accomplished, forexample, via gravitational settling or centrifugation, using, forexample, a settler, decanter centrifuge, disc-stack centrifuge, basketcentrifuge, or hydrocyclone, wherein suitable g-forces and settling orcentrifugation times can be determined using methods known in the art.

In some embodiments of the present disclosure, fermentation products arecrystallized in the fermentation broth prior to removal of cells, celldebris, contaminating salts and various soluble impurities. In many ofthese embodiments, the fermentation product crystals are separated fromfermentation broth, cells, cell debris, contaminating salts and varioussoluble impurities by sedimentation, centrifugation, ultrafiltration,nanofiltration, ion exchange, or any combination thereof.

Lactic Acid Polymers

In certain aspects, the L-lactic acid provided herein, or a salt orderivative thereof is employed as at least one type of polymerizationmaterial to produce a lactic acid polymer. Examples of polymerizationmaterial that is employed, comprise, for example, L-lactic acid, orderivatives (such as lactides), and prepolymers and oligomers resultingfrom polymerizing such monomers to suitable lengths. In someembodiments, the polymerization further comprises D-lactic acid orderivatives (such as lactides) and prepolymers and oligomers thereof.

Non-limiting examples of lactic acid polymers include homopolymers ofL-lactic acid, hetero-block polymers, and various types ofheteropolymers of lactic acid and non-lactic based polymerizationmaterial.

In some embodiments, the lactic acid polymerization materials, or lacticacid polymerization material and another non-lactic acid polymerizationmaterial, can be reacted with a suitable polymerization initiator toproduce lactic acid polymers. Various Lewis acid-metal catalysts such asdioctyl stannate and the likes, and non-nucleophilic Lewis bases, suchas diazabicycloundecane and the likes may be utilized as apolymerization initiator or polymerization catalyst. The polymerizationis performed in solution, in a melt, or in a suspension. See forexample, Garlotta, “A Literature Review of Poly(lactic)acid,” Journal ofPolymers and the Environment, 2001, vol. 9, no. 2, pages 63-84.

EXAMPLES Parent Strain Used in the Examples

The parent strain in Example 1 was a P. kudriavzevii strain auxotrophicfor histidine and uracil due to genetic disruptions in URA2 and HIS3(i.e., the strain cannot grow in media without histidine and uracilsupplementation). Histidine auxotrophy in the parent strain enablesselection of new, engineered strains that carry a HIS3 marker, enablinghistidine prototrophy and indicating desired nucleic acid modification.Likewise, uracil auxotrophy in the parent strain enables selection ofnew, engineered strains that carry a URA2 marker, enabling uracilprototrophy and indicating desired nucleic acid modification. Thus,cells that were successfully modified with exogenous nucleic acids tocomprise desired genetic modifications can grow in media withouthistidine and/or uracil supplementation, dependent on the selectionmarker included in the exogenous nucleic acid. Following confirmation ofcorrect strain engineering, the selection marker(s) were removed by, forexample, homologous recombination and marker loopout. Removing themarker enables subsequent rounds of strain engineering using the sameselection markers.

Media Used in the Examples

Complete supplement mixture (CSM) medium. CSM medium comprised Adenine10 mg/L; L-Arginine HCl 50 mg/L; L-Aspartic Acid 80 mg/L; L-HistidineHCl 20 mg/L; L-Isoleucine 50 mg/L; L-Leucine 100 mg/L; L-Lysine HCl 50mg/L; L-Methionine 20 mg/L; L-Phenylalanine 50 mg/L; L-Threonine 100mg/L; L-Tryptophan 50 mg/L; L-Tyrosine 50 mg/L; Uracil 20 mg/L; L-Valine140 mg/L. The YNB used in the CSM comprised Ammonium sulfate 5.0 g/L,Biotin 2.0 mg/L, Calcium pantothenate 400 mg/L, Folic acid 2.0 mg/L,Inositol 2.0 mg/L, Nicotinic acid 0-400 mg/L, p-Aminobenzoic acid 200mg/L, Pyridoxine HCl 400 mg/L, Riboflavin 200 mg/L, Thiamine HCl 400mg/L, Boric acid 500 mg/L, Copper sulfate 40 mg/L, Potassium iodide 100mg/L, Ferric chloride 200 mg/L, Manganese sulfate 400 mg/L, Sodiummolybdate 200 mg/L, Zinc sulfate 400 mg/L, Potassium phosphate monobasic1.0 g/L, Magnesium sulfate 0.5 g/L, Sodium chloride 0.1 g/L, and Calciumchloride 0.1 g/L.

Complete supplement mixture minus histidine (CSM-His) medium. CSM-Hismedium is identical to CSM medium with the exception that histidine wasnot included in the medium. Engineered strains auxotrophic for histidineare unable to grow on CSM-His medium while engineered strains containingexogenous nucleic acids comprising a histidine selectable marker (forexample, HIS3) are capable of growth in CSM-His medium.

Complete supplement mixture minus uracil (CSM-Ura) medium. CSM-Uramedium is identical to CSM medium with the exception that uracil was notincluded in the medium. Engineered strains auxotrophic for uracil areunable to grow on CSM-Ura medium while engineered strains containingexogenous nucleic acids comprising a uracil selectable marker (forexample, URA2) are capable of growth in CSM-Ura medium.

BM02 medium. BM02 medium is Glucose 125 g/L, K₂SO₄ 0.816 g/L, Na₂SO₄0.1236, MgSO4-7H2O 0.304 g/L, Urea 4.3 g/L, Myo-inositol 2 mg/L, ThiaminHCl 0.4 mg/L, Pyridoxal HCl 0.4 mg/L, Niacin 0.4 mg/L, Ca-Pantothenate0.4 mg/L, Biotin mg/L, Folic acid 2 mg/L, PABA 200 mg/L, Riboflavin 200mg/L, Boric acid 0.25 mg/L, Copper sulfate pentahydrate 393 mg/L, Ironsulfate 11.0 mg/L, Manganese chloride 1.6 mg/L, Sodium molybdate 100mg/L, Zinc sulfate 4 mg/L, and EDTA 11 mg/L.

BM02-P medium. BM02-P medium is BM02 medium with 1 g/L potassiumphosphate.

YPE medium. YPE medium is Bacto peptone 20 g/L, Yeast extract 10 g/L,and Ethanol 2% (v/v).

Example 1: Construction of Recombinant P. Kudriavzevii Strain, LPK15779,with Eliminated Expression of Pyruvate Decarboxylase

Example 1 describes the construction of a pyruvate decarboxylase (PDC)minus P. kudriavzevii, LPK15779, wherein all three PDC genes, i.e.,Pdc1, Pdc5 and Pdc6, were genetically disrupted to eliminate expressionof PkPDC1 (SEQ ID NO: 6), PkPDC5 (SEQ ID NO: 7), and PkPDC6 (SEQ ID NO:8).

The parent P. kudriavzevii strain used in this example was auxotrophicfor uracil and histidine. To eliminate PDC expression, the Pdc1, Pdc5and Pdc6 genes in the P. kudriavzevii genome were disruptedsequentially. The P. kudriavzevii strain was diploid and two copies ofeach pyruvate decarboxylase gene were present at the indicated locus;therefore, disruption of each gene was achieved by deleting of both genecopies.

A URA3 selectable marker, amplified by PCR, was provided to the parentP. kudriavzevii strain to complement the uracil auxotrophic deficiency.The URA3 selectable marker comprised unique upstream and downstreamhomologous regions for homologous recombination at the P. kudriavzeviiPdc1 locus, a transcriptional promoter, a URA3 coding region, and atranscriptional terminator. The transcriptional promoter 5′ of URA3 wasthe P. kudriavzevii TEF1 promoter (pPkTEF1) and the transcriptionalterminator 3′ of URA3 was the S. cerevisiae TDH3 terminator (tScTDH3).The PCR product of the URA3 selectable marker was gel-purified andprovided as exogenous nucleic acids to P. kudriavzevii. Transformationwas carried out in a single step and gene deletion was achieved byhomologous recombination. Transformants were selected on CSM-Ura mediumand successful deletion of both copies of the gene encoding PkPDC1 wasconfirmed by genetic sequencing of this locus and the flanking regions.After successful construction of a recombinant P. kudriavzeviicomprising a Pdc1 genetic disruption, the URA3 selectable marker wasremoved from the recombinant strain genome by recombination and markerloopout.

The URA3 selectable marker and genetic disruption strategy describedabove were reused to next disrupt the Pdc5 and Pdc6 genes in succession.Deletion of the native genes encoding PkPDC5 and PkPDC6 was confirmed bygenetic sequencing of this locus and the flanking regions. The P.kudriavzevii strain that results from Example 1, LPK15779, was withoutany URA3 selectable marker. The URA3 selectable marker was absent in thefollowing examples that describe further strain engineering or strainperformance testing. Thus, Example 1 produces a PDC minus (i.e.,comprises deletion of native genes encoding PkPDC1, PkPDC5, and PkPDC6),uracil and histidine auxotrophic P. kudriavzevii, which was thebackground strain for Example 2 below.

Example 2: Construction of Recombinant P. kudriavzevii BackgroundStrain, LPK15942, with Eliminated Expression of Pyruvate Decarboxylaseand Pyruvate Dehydrogenase Complex

Example 2 describes the construction of a pyruvate dehydrogenase complex(PDH) minus P. kudriavzevii, LPK15942, wherein expression of PDH waseliminated via genetic disruption of the Pda1 gene. Pda1 encodes for theE1 a-subunit (PkPDA1; SEQ ID NO: 9) of the PDH. When PkPDA1 expressionis eliminated, PDH cannot assemble into a functional complex. Thus, PDHexpression is also eliminated and the recombinant host cell is unable tocatalyze the conversion of pyruvate, coenzyme A and NAD+ to acetyl-CoA,CO₂ and NADH in the host cell mitochondria. This genetic disruption hasthe end result of decreasing respiration, thereby decreasing formationof byproduct CO₂ and increasing L-lactic acid production.

PkPDA1 was genetically disrupted using the same engineering strategy asdescribed above in Example 1. LPK15779, a PDC minus, uracil andhistidine auxotrophic P. kudriavzevii strain from Example 1 was thebackground strain used in Example 2.

A HIS3 selectable marker, amplified by PCR, was provided to thebackground strain (from Example 1) to complement the histidineauxotophic deficiency. The HIS3 selectable marker comprised uniqueupstream and downstream homologous regions for homologous recombinationat the Pda1 locus of the background strain genome, a transcriptionalpromoter, a HIS3 coding region, and a transcriptional terminator. Thetranscriptional promoter 5′ of HIS3 was the P. kudriavzevii TEF1promoter (pPkTEF1) and the transcriptional terminator 3′ of HIS3 was theS. cerevisiae TDH3 terminator (tScTDH3). The PCR product of the HIS3selectable marker was gel-purified and provided as exogenous nucleicacids to the background strain. Transformation was carried out in asingle step and gene deletion was achieved by homologous recombination.Transformants were selected on CSM-His medium and successful deletion ofboth copies of the genes encoding PkPDA1 was confirmed by geneticsequencing of this locus and the flanking regions. After successfulconstruction of a recombinant P. kudriavzevii comprising a Pda1 geneticdisruption, the HIS3 selectable marker was removed from the recombinantstrain genome by recombination and marker loopout.

The P. kudriavzevii strain that resulted from Example 2, LPK15942, waswithout a HIS3 selectable marker. The HIS3 selectable marker was absentin the following examples that describe further strain engineering orstrain performance testing. Example 2 produced a PDC minus, PDH minus,uracil and histidine auxotrophic P. kudriavzevii (i.e., the straincomprised deletion of native genes encoding PkPDC1, PkPDC5, PkPDC6, andPkPDA1), which was the background strain used in Example 3.

Example 3: Construction of Recombinant P. kudriavzevii Strain,LPK151316, with Eliminated Expression of the Mitochondrial External NADHDehydrogenase

Example 3 describes the construction of a mitochondrial external NADHdehydrogenase (NDE1) minus P. kudriavzevii, LPK151316, whereinexpression of NDE1 was eliminated via genetic disruption of the Nde1gene. When PkNDE1 (SEQ ID NO: 11) expression is eliminated, therecombinant host cell is unable to oxidize cytosolic NAD(P)H to NAD(P)+,thus not competing with the L-lactic acid pathway for NAD(P)H, which isutilized by the L-lactic acid pathway to make L-lactic acid. Thisgenetic disruption has the end result of increased L-lactic acid productformation.

PkNDE1 was genetically disrupted using the same engineering strategy asdescribed in Examples 1 and 2. LPK15942, a PDC minus, PDH minus, anduracil auxotrophic P. kudriavzevii from Example 2 was the backgroundstrain used in Example 3.

A HIS3 selectable marker, amplified by PCR, was provided to thebackground strain (from Example 2) to complement the histidineauxotrophic deficiency. The HIS 3 selectable marker comprised uniqueupstream and downstream homologous regions for homologous recombinationat the Pda1 locus of the background strain genome, a transcriptionalpromoter, a HIS3 coding region, and a transcriptional terminator. Thetranscriptional promoter 5′ of HIS3 was the P. kudriavzevii TEF1promoter (pPkTEF1) and the transcriptional terminator 3′ of HIS3 was theS. cerevisiae TDH3 terminator (tScTDH3). The PCR product of the HIS3selectable marker was gel-purified and provided as exogenous nucleicacids to the background strain. Transformation was carried out in asingle step and gene deletion was achieved by homologous recombination.Transformants were selected on CSM-His medium and successful deletion ofboth copies of genes encoding PkNDE1 was confirmed by genetic sequencingof this locus and the flanking regions. After successful construction ofa recombinant P. kudriavzevii comprising a Nde1 genetic disruption, theHIS3 selectable marker was removed from the recombinant strain genome byrecombination and marker loopout.

The P. kudriavzevii strain that resulted from Example 3, LPK151316, waswithout a HIS3 selectable marker. The HIS selectable marker was absentin the following examples that describe further strain engineering orstrain performance testing. Example 3 produced a PDC minus, PDH minus,NDE1 minus, uracil and histidine auxotrophic P. kudriavzevii (i.e., thestrain comprised deletion of native genes encoding PkPDC1, PkPDC5,PkPDC6, PkPDA1, and PkNDE1), which was the background strain used inExample 5.

Example 4: Recombinant P. kudriavzevii Background Strain, LPK151316,does not Naturally Produce L-Lactic Acid

Example 4 describes the culturing and analysis of LPK151316 (fromExample 3) for L-lactic acid production before LPK151316 was used as thebackground strain for genomic integration of the L-lactic acid pathway(Example 5). LPK151316 colonies are used to inoculate replicate tubes of15 mL of YPE medium and are incubated at 30° C. with 80% humidity andshaking at 250 rpm for 20 hours. These replicate tubes of pre-culturesare used to inoculate baffled flask replicates of 250 mL of BM02-P mediawith 10% glucose, 1% ethanol and 40 g/L CaCO₃. Pre-cultures are diluted50× with 1 M HCl for OD600 measurements to inform appropriate dilutionof pre-cultures to produce a starting culture biomass of 1 g/L dry cellweight (DCW). Baffled flask cultures are then incubated at 30° C. with80% humidity and shaking at 250 rpm. After 48 hours, the cultures arediluted 10× with 12 M HCl, spin-filtered and frozen for storage. Samplesare analyzed by HPLC within 48 hours of harvest.

For HPLC analysis, frozen samples are thawed analyzed by HPLC using aBio-Rad Aminex 87H column (300×7.8 mm) and a Bio-Rad FermentationMonitoring column (#1250115; 150×7.8 mm) installed in series, with anisocratic elution rate of 0.8 mL/min with water at pH 1.95 (withsulfuric acid) at 30° C. Refractive index and UV 210 nm measurements areacquired for 35 minutes.

The LPK151316 background strain does not produce detectable amounts ofL-lactic acid. Thus, all engineered P. kudriavzevii strains built fromthis background strain are incapable of producing L-lactic acid withoutthe heterologous nucleic acids that encode the L-lactic acid pathway(Example 5).

Example 5: Construction of Recombinant P. kudriavzevii Strains LPK15980,LPK15982, LPK15985, LPK15987, LPK15989, and LPK15990 Comprising anEnzyme that Converts Pyruvate to L-Lactic Acid

Example 5 describes the construction of recombinant P. kudriavzevii hostcells of the present disclosure wherein each strain comprisedheterologous nucleic acids encoding an enzyme of the L-lactic acidpathway capable of carrying out the activity of the LLDH. LPK15980comprised the LLDH from Bacillus megaterium (abbv. BmLDH; UniProt ID:P00345). LPK15982 comprised the LLDH from Bostaurus (abbv. BtLDH;UniProt ID: P19858). LPK15985 comprised the LLDH from Lactobacilluscasei (abbv. LcLDH; UniProt ID: P00343). LPK15987 comprised the LLDHfrom Lactobacillus helveticus (abbv. LhLDH; UniProt ID: 032765).LPK15989 comprised the LLDH from Lactobacillus plantarum (abbv. LhLDH;UniProt ID: P56512). LPK15990 comprised the LLDH from Pediococcusacidilactici (abbv. LhLDH; UniProt ID: Q59645). In each strain,insertion of the heterologous nucleic acids encoding the LLDHgenetically disrupts both copies of PkADH6C, i.e., producing an ADH6Cminus phenotype.

The PkPDC1, PkPDC5, PkPDC6, and uracil and histidine auxotrophic P.kudriavzevii, LPK15779 from Example 1 was the background strain used inthis example.

The heterologous nucleic acids used in this example were codon-optimizedfor yeast and were synthesized and provided by Twist Bioscience; eachgene was cloned into its own entry vector, pEV, along with an upstreamtranscriptional promoter and a downstream transcriptional terminator.The transcriptional terminators cloned in from (5′) of each gene wereconstitutive and derived from P. kudriavzevii. The transcriptionalterminators cloned behind (3′) of each gene were derived from S.cerevisiae. The promoter and terminator were the P. kudriavzevii TDH1promoter (pPkTDH1) and the S. cerevisiae TEF1 terminator (tScTEF1),respectively. Additionally, a HIS3 marker was included in theheterologous expression cassette to complement the histidine auxotrophicdeficiency in HIS3 marker comprised a transcriptional promoter, a HIS3coding region, and a transcriptional terminator. The transcriptionalpromoter 5′ of HIS3 was the P. kudriavzevii TEF1 promoter (pPkTEF1) andthe transcriptional terminator 3′ of HIS3 was the S. cerevisiae TDH3terminator (tScTDH3).

All PCR products were purified and provided as exogenous nucleic acidsto P. kudriavzevii. Transformation was carried out in a single step.Transformants were selected on CSM-His medium. Successful integration ofall heterologous nucleic acids encoding the L-lactic acid pathwayenzymes as well as deletion of both copies of the genes encoding PkADH6Cwere confirmed by genetic sequencing of this locus and the flankingregions.

Example 5 produced recombinant host cells that comprised heterologousnucleic acids encoding an enzyme of the L-lactic acid pathway derivedfrom either B. megaterium, B. taurus, L. casei, L. helveticus, L.plantarum, or P. acidilacti. The recombinant host cells furthercomprised genetic disruption of PkPDC1, PkPDC5, PkPDC6, and PkADH6C. Theresulting strains were additionally auxotrophic for uracil.

Example 6: Recombinant P. kudriavzevii Strains LPK15980, LPK15982,LPK15985, LPK15987, LPK15989, and LPK15990 Produce Increased Amounts ofL-Lactic Acid

Example 6 describes the culturing and analysis of recombinant host cellsLPK15980, LPK15982, LPK15985, LPK15987, LPK15989, and LPK15990 fromExample 5 and the control, parental host cell LPK15779. The recombinantstrains were cultured and analyzed by HPLC according to methodsdescribed above in Example 4.

The recombinant strains with an L-lactic acid pathway produced between2-22 g/L L-lactic acid as compared to the background strain LPK15779which did not produce L-lactic acid. Strains LPK15980, LPK15985, andLPK15987, specifically, produced between 13-22 g/L lactic acid.LPK15987, expressing LhLDH, produced the highest lactic acid titer (21.5g/L). This example demonstrates, in accordance with the presentdisclosure, the expression of heterologous nucleic acids encoding anL-lactic acid pathway in recombinant P. kudriavzevii with increasedL-lactic acid yields as compared to a host cell lacking the heterologousL-lactic acid pathway but otherwise genetically identical. This exampledemonstrates, in accordance with the present disclosure, the expressionof heterologous nucleic acids encoding an L-lactic acid pathway inrecombinant P. kudriavzevii can increase L-lactic acid yields ascompared to a host cell lacking the heterologous L-lactic acid pathwaybut is otherwise genetically identical.

Example 7: Construction of Recombinant P. kudriavzevii Strain LPK151048,with Eliminated Expression of Pyruvate Decarboxylase, PyruvateDehydrogenase Complex, and Comprises an Enzyme that Converts Pyruvate toL-Lactic Acid

Example 7 describes the construction of a pyruvate decarboxylase (PDC)minus and pyruvate dehydrogenase complex minus, P. kudriavzevii strain,LPK151048, comprising heterologous nucleic acids encoding LhLDH, anenzyme of the lactic acid pathway capable of converting pyruvate tolactic acid (see Example 6).

P. kudriavzevii strain LPK15942 from Example 3 was the background strainused in this example.

The heterologous nucleic acids used in this example were codon-optimizedfor yeast and were synthesized and provided by Twist Bioscience; eachgene was cloned into its own entry vector, pEV, along with an upstreamtranscriptional promoter and a downstream transcriptional terminator.The transcriptional terminators cloned in from (5′) of each gene wereconstitutive and derived from P. kudriavzevii. The transcriptionalterminators cloned behind (3′) of each gene were derived from S.cerevisiae. The promoter and terminator were the P. kudriavzevii TDH1promoter (pPkTDH1) and the S. cerevisiae TEF1 terminator (tScTEF1),respectively. Additionally, a HIS3 marker was included in theheterologous expression cassette to complement the histidine auxotrophicdeficiency in HIS3 marker comprised a transcriptional promoter, a HIS3coding region, and a transcriptional terminator. The transcriptionalpromoter 5′ of HIS3 was the P. kudriavzevii TEF1 promoter (pPkTEF1) andthe transcriptional terminator 3′ of HIS3 was the S. cerevisiae TDH3terminator (tScTDH3).

All PCR products were purified and provided as exogenous nucleic acidsto P. kudriavzevii. Transformation was carried out in a single step.Transformants were selected on CSM-His medium. Successful integration ofall heterologous nucleic acids encoding the L-lactic acid pathwayenzymes as well as deletion of both copies of the genes encoding PkADH6Cwere confirmed by genetic sequencing of this locus and the flankingregions. After successful construction of recombinant P. kudriavzeviistrain LPK151048, the HIS3 selectable marker was removed from therecombinant strain genome by recombination and marker loopout,generating recombinant P. kudriavzevii strain LPK151205.

Example 7 produced recombinant host cells that comprised heterologousnucleic acids encoding an enzyme of the L-lactic acid pathway derivedfrom Lactobacillus helveticus. The recombinant host cells furthercomprised genetic disruption of PkPDC1, PkPDC5, PkPDC6, PkPDA1, andPkADH6C. The resulting strains were additionally auxotrophic for uracil.

Example 8: Construction of Recombinant P. kudriavzevii Strain LPK151296,with Eliminated Expression of Pyruvate Decarboxylase, PyruvateDehydrogenase Complex, and External Mitochondrial NADH Dehydrogenase,and Comprises an Enzyme that Converts Pyruvate to L-Lactic Acid

Example 8 describes the construction of a pyruvate decarboxylase (PDC)minus, pyruvate dehydrogenase complex (PDA1) minus, externalmitochondrial NADH dehydrogenase (NDE1) minus P. kudriavzevii strain,LPK151296, comprising heterologous nucleic acids encoding LhLDH, anenzyme of the lactic acid pathway capable of converting pyruvate tolactic acid (see Example 6).

P. kudriavzevii strain LPK151205 from Example 7 was the backgroundstrain used in this example. PkNDE1 was genetically disrupted using thesame engineering strategy as described in Example 3. Transformants wereselected on CSM-His medium and successful deletion of both copies ofgenes encoding PkNDE1 was confirmed by genetic sequencing of this locusand the flanking regions.

Example 8 produced recombinant host cells that comprised heterologousnucleic acids encoding an enzyme of the L-lactic acid pathway derivedfrom Lactobacillus helveticus. The recombinant host cells furthercomprised genetic disruption of PkPDC1, PkPDC5, PkPDC6, PkPDA1, PkNDE1,and PkADH6C. The resulting strains were additionally auxotrophic foruracil.

Example 9: Recombinant P. kudriavzevii Strain LPK15296 Produces anIncreased Amount of L-Lactic Acid as Compared to P. kudriavzevii StrainLPK151048

Example 9 describes the culturing and analysis of recombinant host cellsLPK151296 and LPK151048 from Examples 8 and 7, respectively. LPK151296is genetically identical to LPK151048 with the exception that itadditional comprises deletion of PkNDE1, encoding a mitochondrialexternal NADH dehydrogenase. The recombinant strains were cultured andanalyzed by HPLC according to methods described above in Example 4.

Recombinant P. kudriavzevii LPK151296 produced 77+/−1.5 g/L L-lacticacid while recombinant P. kudriavzevii LPK151048 produced 36+/−1.2 g/LL-lactic acid (mean+/−standard deviation). This example demonstrates, inaccordance with the present disclosure, that deletion or disruption of amitochondrial external NADH dehydrogenase in recombinant P. kudriavzeviicomprising heterologous nucleic acids encoding an L-lactic acid pathwayenzyme increases L-lactic acid production as compared to a host cellexpressing a mitochondrial external NADH dehydrogenase but otherwisegenetically identical.

Lactobacillus helveticus SEQ ID NO: 1MAREEKPRKV ILVGDGAVGS TFAFSMVQQG IAEELGIIDI AKEHVEGDAI DLADATPWTS 60PKNIYAADYP DCKDADLVVI TAGAPQKPGE TRLDLVNKNL KILSSIVEPV VESGFEGIFL 120VVANPVDILT HATWRMSGFP KDRVIGSGTS LDTGRLQKVI GKMENVDPSS VNAYMLGEHG 180DTEFPAWSYN NVAGVKVADW VKAHNMPESK LEDIHQEVKD MAYDIINKKG ATFYGIGTAS 240AMIAKAILND EHRVLPLSVP MDGEYGLHDL HIGTPAVVGR KGLEQVIEMP LSDKEQELMT 300Saccharomyces cerevisiae SEQ ID NO: 2MSSTDEHIEK DISSRSNHDD DYANSVQSYA ASEGQVDNED LAATSQLSRH LSNILSNEEG 60IERLESMARV ISHKTKKEMD SFEINDLDFD LRSLLHYLRS RQLEQGIEPG DSGIAFKNLT 120AVGVDASAAY GPSVEEMFRN IASIPAHLIS KFTKKSDVPL RNIIQNCTGV VESGEMLFVV 180GRPGAGCSTF LKCLSGETSE LVDVQGEFSY DGLDQSEMMS KYKGYVIYCP ELDFHFPKIT 240VKETIDFALK CKTPRVRIDK MTRKQYVDNI RDMWCTVFGL RHTYATKVGN DFVRGVSGGE 300RKRVSLVEAQ AMNASIYSWD NATRGLDAST ALEFAQAIRT ATNMVNNSAI VAIYQAGENI 360YELFDKTTVL YNGRQIYFGP ADKAVGYFQR MGWVKPNRMT SAEFLTSVTV DFENRTLDIK 420PGYEDKVPKS SSEFEEYWLN SEDYQELLRT YDDYQSRHPV NETRDRLDVA KKQRLQQGQR 480ENSQYVVNYW TQVYYCMIRG FQRVKGDSTY TKVYLSSFLI KALIIGSMFH KIDDKSQSTT 540AGAYSRGGML FYVLLFASVT SLAEIGNSFS SRPVIVKHKS YSMYHLSAES LQEIITEFPT 600KFVAIVILCL ITYWIPFMKY EAGAFFQYIL YLLTVQQCTS FIFKFVATMS KSGVDAHAVG 660GLWVLMLCVY AGFVLPIGEM HHWIRWLHFI NPLTYAFESL VSTEFHHREM LCSALVPSGP 720GYEGISIANQ VCDAAGAVKG NLYVSGDSYI LHQYHFAYKH AWRNWGVNIV WTFGYIVFNV 780ILSEYLKPVE GGGDLLLYKR GHMPELGTEN ADARTASREE MMEALNGPNV DLEKVIAEKD 840VFTWNHLDYT IPYDGATRKL LSDVFGYVKP GKMTALMGES GAGKTTLLNV LAQRINMGVI 900TGDMLVNAKP LPASFNRSCG YVAQADNHMA ELSVRESLRF AAELRQQSSV PLEEKYEYVE 960KIITLLGMQN YAEALVGKTG RGLNVEQRKK LSIGVELVAK PSLLLFLDEP TSGLDSQSAW 1020SIVQFMRALA DSGQSILCTI HQPSATLFEQ FDRLLLLKKG GKMVYFGDIG PNSETLLKYF 1080ERQSGMKCGV SENPAEYILN CIGAGATASV NSDWHDLWLA SPECAAARAE VEELHRTLPG 1140RAVNDDPELA TRFAASYMTQ IKCVLRRTAL QFWRSPVYIR AKFFECVACA LFVGLSYVGV 1200NHSVGGAIEA FSSIFMLLLI ALAMINQLHV FAYDSRELYE VREAASNTFH WSVLLLCHAA 1260VENFWSTLCQ FMCFICYYWP AQFSGRASHA GFFFFFYVLI FPLYFVTYGL WILYMSPDVP 1320SASMINSNLF AAMLLFCGIL QPREKMPAFW RRLMYNVSPF TYVVQALVTP LVHNKKVVCN 1380PHEYNIMDPP SGKTCGEFLS TYMDNNTGYL VNPTATENCQ YCPYTVQDQV VAKYNVKWDH 1440RWRNFGFMWA YICFNIAAML ICYYVVRVKV WSLKSVLNFK KWFNGPRKER HEKDTNIFQT 1500VPGDENKITK K 1511 Saccharomyces cerevisiae SEQ ID NO: 3MDTQIAITGV AVGKEINNDN SKTDQKVSLP KADVPCIDKA TQTIIEGCSK DDPRLSYPTK 60LETTEKGKTK RNSFACVCCH SLKQKCEPSD VNDIYRKPCR RCLKHKKLCK FDLSKRTRKR 120KPRSRSPTPF ESPMVNVSTK SKGPTDSEES SLKDGTSYLA SFPSDPNAKQ FPNSRTVLPG 180LQQSLSDLWS TLSQPPSYGA REAETTSTGE ITTNNHTKSN GSVPTNPAVL ASNDEHTNIS 240DAPVIYSTYN SPVPISSAPT SINSEALFKH RPKIVGDEET QNVKVKRQKK SYSRHMTRSF 300RKQLQSLIIS QKGKIRDISM KLDTWSKQWN DLVEKSMFLP TIADPVSVGI ISHEEATLRL 360HLYKTEISYL SKLPFIKVEE NVSVDELRKK KPILFSVIMS CVSIVLTPKQ TTRGTIMKLD 420SFVLNLITNQ IFKANNKSIE IIESLSTLCL WYNFFEWSSK TRYHIFNYIC CCLTRDLGPT 480YVNRSFGMFS DEDPKRFKSP LELYSNGASL TLLVYISALN ISIFLRQSIQ ARWSHVTEKA 540CEDLVKETKK SRHYDNDKLL LDSADDPILV QFAKMNHVLE NIHTHLHERD LNDDEFDDPI 600FTKKYLNKLM EKYHKQLQEI FTKLDRNRPR VIAFYYSVEA YLYQYKLAVF IGEMSHTINE 660KVELPREIMD DFVKCYHCCK SALEEFSKLE PILITSLPLF HTSRIIYTVG MLLLKLRYSV 720VAIPSFHDLM PLTDDAIALV IGVNNLLEKT SELYPFNNSL YKFRYVIALF CQTYANKVID 780VADRYNAERE KLKEKQVIDE VSNGHDGTKP INAYVTESQK MPTEEDPIID NNTNQNITAV 840PDEMLPVYSR VRDDTAAMNL NINSTSYMNE SPHEHRESMT GTTLLPPPFI SNDVTNSADS 900TNIKPSPSSS VDNLNDYLTD INSLAWGVNS LNDEFWTDLF MNDI 944Schizosaccharomyces pombe SEQ ID NO: 4MGELKEILKQ RYHELLDWNV KAPHVPLSQR LKHFTWSWFA CTMATGGVGL IIGSFPFRFY 60GLNTIGKIVY ILQIFLFSLF GSCMLFRFIK YPSTIKDSWN HHLEKLFIAT CLLSISTFID 120MLAIYAYPDT GEWMVWVIRI LYYIYVAVSF IYCVMAFFTI FNNHVYTIET ASPAWILPIF 180PPMICGVIAG AVNSTQPAHQ LKNMVIFGIL FQGLGFWVYL LLFAVNVLRF FTVGLAKPQD 240RPGMFMFVGP PAFSGLALIN IARGAMGSRP YIFVGANSSE YLGFVSTFMA IFIWGLAAWC 300YCLAMVSFLA GFFTRAPLKF ACGWFAFIFP NVGFVNCTIE IGKMIDSKAF QMFGHIIGVI 360LCIQWILLMY LMVRAFLVND LCYPGKDEDA HPPPKPNTGV LNPTFPPEKA PASLEKVDTH 420VTSTGGESDP PSSEHESV 438 Kluyveromyces marxianus SEQ ID NO: 5MSNSSSSEGK TNEDGRNSVH SSDSFAQSVA SFHLDDNESQ NVTAQLSQQI TNVLSNSNGA 60ERIESLARVI STKTKKQMES FEVNQLDFDL KALLNYLRSS QLEQGIEPGD SGIAFHDLTA 120VGIDASAAFG PSVEEMVRSW IHFPVRLWKK ICRQKSETPL RNIIQHCTGV VESGEMLFVV 180GRPGAGCSTL LKCLSGETGE LVEVTGDISY DGLSQEEMMQ KFKGYVIYCP ELDFHFPKIT 240VKETIDFALK CKTPRSRIDH LTRAQYVDNM RDLWCTVFGL THTYATNVGN DVVRGVSGGE 300RKRVSLVEAL AMNASIYSWD NATRGLDAST ALEFAQAIRT ATNMMNNSAI VAIYQAGENI 360YQLFDKTTVL YNGKQVYFGP ADEAVGYFER MGYIKPNRMT SAEFLTSATV DFENRTLEVR 420EGYEEKIPKS STEMEAYWHN SPEYAKATEL FNEYCQSHPE EETRQRLETA KKQRLQKGQR 480EKSQFVVTFW AQVWYCMIRG FQRVKGDSTY TKVYLSSFLT KGLIVGSMFH KIDPKSQSTT 540EGAYSRGGLL FYVLLFAALT SLAEISNSFQ NRAIIVKQKT YSMYHTSAES LQEIFTEIPT 600KFVAILTLSL VSYWIPVLKY DAGSFFQYLL YLFTTQQCTS FIFKLVATLT KDGGTAHAIG 660GLWVLMLTVY AGFVLPIGNM HHWIRWFHYL NPLTYAYESL MSTEFHGRKM LCSRLLPSGP 720GYENVSIAHK ICDAAGAVAG QLYVSGDAYV LKKYHFRYKH AWRDWGINIV WTFGYIVMNV 780VMSEYLKPLE GGGDLLLYKR GHMPELGSES VDSKVASREE MMESLNGPGV DLEKVIASKD 840VFTWNHLNYT IPYDGATRQL LSDVFGYVKP GKMTALMGES GAGKTTLLNV LAQRINVGVI 900TGDMLVNAKP LPPSFNRSCG YVAQADNHMG ELSVRESLRF AAELRQPKSV PLQEKYDYVE 960KIISLLGMEK YAEAIIGKTG RGLNVEQRKK LSIGVELVAK PSLLLFLDEP TSGLDSQSAW 1020SIVQFMRALA DSGQSILCTI HQPSATLFEQ FDRLLLLKKG GKMVYFGDIG ENSSTLLNYF 1080ERQSGVKCGK SENPAEYMLN CIGAGATASA DADWHDLWLQ SPECAAAREE VEELHRTLAS 1140RPVTDDKELA GRYAASYLTQ MKCVFRRTNI QFWRSPVYIR AKFLECVLCA LFVGLSYVGV 1200DHSIAGASQS FSSIFMMLLI ALAMVNQLHV FALDSRELYE VREAASNTFH WSVLLLNHTF 1260VEIIWSTLCE FICWICYYWP AQYSGRASHA GYFFLIYVIM FPAYFVSYGC WVFYMSPDVP 1320SASMINSNLF AGMLLFCGIL QPKDKMPGFW KRFMYNVSPF TYVVQSLVTP LVQGKKVRCT 1380KNEFAVVNPP EGQTCSQYFA RFIKDNTGYL KNPNDTESCH YCPYSYQQEV VEQYNVRWVY 1440RWRNFGFLWA YIGFNFFAML ACYWVLRVKN YSITSIFGVF KIGNWKKAIH HDSRHEKDHT 1500IFQEKPGDAA NVQKTKA 1517 Pichia kudriavzevii SEQ ID NO: 6MTDKISLGTY LFEKLKEAGS YSIFGVPGDF NLALLDHVKE VEGIRWVGNA NELNAGYEAD 60GYARINGFAS LITTFGVGEL SAVNAIAGSY AEHVPLIHIV GMPSLSAMKN NLLLHHTLGD 120TRFDNFTEMS KKISAKVEIV YDLESAPKLI NNLIETAYHT KRPVYLGLPS NFADELVPAA 180LVKENKLHLE EPLNNPVAEE EFIHNVVEMV KKAEKPIILV DACAARHNIS KEVRELAKLT 240KFPVFTTPMG KSTVDEDDEE FFGLYLGSLS APDVKDIVGP TDCILSLGGL PSDFNTGSFS 300YGYTTKNVVE FHSNYCKFKS ATYENLMMKG AVQRLISELK NIKYSNVSTL SPPKSKFAYE 360SAKVAPEGII TQDYLWKRLS YFLKPRDIIV TETGTSSFGV LATHLPRDSK SISQVLWGSI 420GFSLPAAVGA AFAAEDAHKQ TGEQERRTVL FIGDGSLQLT VQSISDAARW NIKPYIFILN 480NRGYTIEKLI HGRHEDYNQI QPWDHQLLLK LFADKTQYEN HVVKSAKDLD ALMKDEAFNK 540EDKIRVIELF LDEFDAPEIL VAQAKLSDEI NSKAA 575 Pichia kudriavzeviiSEQ ID NO: 7MLQTANSEVP NASQITIDAA SGLPADRVLP NITNTEITIS EYIFYRILQL GVRSVFGVPG 60DFNLRFLEHI YDVHGLNWIG CCNELNAAYA ADAYAKASKK MGVLLTTYGV GELSALNGVA 120GAYTEFAPVL HLVGTSALKF KRNPRTLNLH HLAGDKKTFK KSDHYKYERI ASEFSVDSAS 180IEDDPIEACE MIDRVIYSTW RESRPGYIFL PCDLSEMKVD AQRLASPIEL TYRFNSPVSR 240VEGVADQILQ LIYQNKNVSI IVDGFIRKFR MESEFYDIME KFGDKVNIFS TMYGKGLIGE 300EHPRFVGTYF GKYEKAVGNL LEASDLIIHF GNFDHELNMG GFTFNIPQEK YIDLSAQYVD 360ITGNLDESIT MMEVLPVLAS KLDSSRVNVA DKFEKFDKYY ETPDYQREAS LQETDIMQSL 420NENLTGDDIL IVETCSFLFA VPDLKVKQHT NIILQAYWAS IGYALPATLG ASLAIRDFNL 480SGKVYTIEGD GSAQMSLQEL SSMLRYNIDA TMILLNNSGY TIERVIVGPH SSYNDINTNW 540QWTDLLRAFG DVANEKSVSY TIKEREQLLN ILSDPSFKHN GKFRLLECVL PMFDVPKKLG 600QFTGKIPA 608 Pichia kudriavzevii SEQ ID NO: 8MAPVSLETCT LEFSCKLPLS EYIFRRIASL GIHNIFGVPG DYNLSFLEHL YSVPELSWVG 60CCNELNSAYA TDGYSRTIGH DKFGVLLTTQ GVGELSAANA IAGSFAEHVP ILHIVGTTPY 120SLKHKGSHHH HLINGVSTRE PTNHYAYEEM SKNISCKILS LSDDLTNAAN EIDDLFRTIL 180MLKKPGYLYI PCDLVNVEID ASNLQSVPAN KLRERVPSTD SQTIAKITST IVDKLLSSSN 240PVVLCDILTD RYGMTAYAQD LVDSLKVPCC NSFMGKALLN ESKEHYIGDF NGEESNKMVH 300SYISNTDCFL HIGDYYNEIN SGHWSLYNGI NKESIVILNP EYVKIGSQTY QNVSFEDILP 360AILSSIKANP NLPCFHIPKI MSTIEQIPSN TPISQTLMLE KLQSFLKPND VLVTETCSLM 420FGLPDIRMPE NSKVIGQHFY LSIGMALPCS FGVSVALNEL KKDSRLILIE GDGSAQMTVQ 480ELSNFNRENV VKPLIILLNN SGYTVERVIK GPKREYNDIR PDWKWTQLLQ TFGMDDAKSM 540KVTTPEELDD ALDEYGNNLS TPRLLEVVLD KLDVPWRFNK MVGN 584 Pichia kudriavzeviiSEQ ID NO: 9MLRLFSRRTP SVRALPKFTR SLATASPEAG AQEVSNLHDI VEIELPEYSF EGYKLDVPEL 60NYSTEKGTLL QMYKDMVIIR RMEMAADALY KAKKIRGFCH LSVGQEAIAV GIENAITKQD 120DIITSYRCHG TTYMRGASVQ EVLAELMGRR SGVSYGKGGS MHMYTKGFYG GNGIVGAQVP 180LGTGLAFAHH YRDQKNMTWT MYGDGAANQG QVFESFNMAK LWNLPCVFTC ENNKYGMGTS 240ASRSSAMTEY YKRGQYIPGL KVNGMDILAV YQAAKFAKEW TSNDNGPLVI EFETYRYGGH 300SMSDPGTTYR TREEVQNMRS KKDPIAGLKA HLLEFNIATE EEIKAFDKSA RKYVDEQVKL 360ADASPPPEAK MSILFEDVYV PGSEIPVLRG RIRDDSWSFE KGGFAYK 407Pichia kudriavzevii SEQ ID NO: 10MVSPAERLST IASTIKPNRK DSTSLQPEDY PEHPFKVTVV GSGNWGCTIA KVIAENTVER 60PRQFQRDVNM WVYEELIEGE KLTEIINTKH ENVKYLPGIK LPVNVVAVPD IVEACAGSDL 120IVFNIPHQFL PRILSQLKGK VNPKARAISC LKGLDVNPNG CKLLSTVITE ELGIYCGALS 180GANLAPEVAQ CKWSETTVAY TIPDDFRGKG KDIDHQILKS LFHRPYFHVR VISDVAGISI 240AGALKNVVAM AAGFVEGLGW GDNAKAAVMR IGLVETIQFA KTFFDGCHAA TFTHESAGVA 300DLITTCAGGR NVRVGRYMAQ HSVSATEAEE KLLNGQSCQG IHTTREVYEF LSNMGRTDEF 360PLFTTTYRII YENFPIEKLP ECLEPVED 388 Pichia kudriavzevii SEQ ID NO: 11MIPRLNPLLN ISHLRGGPKF IGKAIKPSQF EFRKNNFRFN STSTKTGSAR TIKSGFLSWS 60FRAATFTGIA GWLYLTYLVY KETNPGSQSP QTEFSEIGNK KKNIVILGSG WGAVSVLKTL 120DTTKYNVTIV SPRNYFLFTP LLPSVPSGTI DIKSICDSIR TIARQTPGEV TYLEAAATDI 180DPVKKTIKLE HKSQRFLIGD AFTSEGDVIE NELSYDYLVY AVGATVNTFG IPGIPEYASY 240LKEANDATAV RQKLFNQIEA SRLLPKDSED RKRLLSFVVC GGGPTGVELA AEIKDYIDQD 300LCKFIPGIEK EMQVTLIEAQ HNVLSMFHPK LIEYTKEVFK QQNLHLQVDT MVKKVDDKNV 360YATYRHPDGK TEDMVIPYGT LVWAGGNAQR KLTRDLSSKI IEQKTARRGL LVDEYLKLDG 420DDSIYAIGDC TFTPNPPTAQ VAHQQGEYLG EHFNKLAKID ELNYLITNST DDSTKYSKRL 480ERAEKAIKPF EYDHQGALAY VGSERAVADL HWGSWSTVAL GGTMTFFFWR TAYVSMLLSI 540RNKILVVTDW VKVAIFGRDC SQE

1. A recombinant cell comprising: a heterologous nucleic acid encodingL-lactate dehydrogenase having at least 60% amino acid identity with SEQID NO: 1; wherein the heterologous nucleic acid is expressed in asufficient amount to produce L-lactic acid.
 2. The recombinant cell ofclaim 1, wherein the L-lactate dehydrogenase has at least 65% amino acididentity with SEQ ID NO:
 1. 3. The recombinant cell of claim 1, whereinthe L-lactate dehydrogenase has at least 70% amino acid identity withSEQ ID NO:
 1. 4. The recombinant cell of claim 1, wherein the L-lactatedehydrogenase has at least 75% amino acid identity with SEQ ID NO: 1.5-9. (canceled)
 10. The recombinant cell of claim 1, wherein theL-lactate dehydrogenase is SEQ ID NO:
 1. 11. The recombinant cell ofclaim 1, wherein the cell is of the genera Candida and does not expressa Crabtree-negative phenotype.
 12. The recombinant cell of claim 1,wherein the cell is not of the genera Candida and expresses aCrabtree-negative phenotype.
 13. The recombinant cell of claim 1,wherein, the cell is a yeast cell.
 14. The recombinant cell of claim 13,wherein the yeast cell does not have a deletion or disruption of anative L- or D-lactate:ferricytochrome c oxidoreductase gene.
 15. Therecombinant cell of claim 13, which is selected from Pichia kudriavzeviiand Saccharomyces cerevisiae. 16-17. (canceled)
 18. The recombinant cellof claim 1, wherein: the L-lactate dehydrogenase comprises the residuesGXGXXG, wherein X refers to any amino acid; and 1-3 amino acid residueslocated 20-22 residues downstream from the GXGXXG sequence is mutated toa neutral amino acid.
 19. The recombinant cell of claim 1, wherein: theL-lactate dehydrogenase comprises the residues GXGXXG, wherein X refersto any amino acid; and a negatively charged amino acid located 18-20residues downstream from the GXGXXG sequence is mutated to a neutralamino acid.
 20. The recombinant cell of claim 1, further comprising oneor more heterologous nucleic acids encoding one or more proteinsselected from organic acid transporters and redox cofactor biogenesisproteins.
 21. The recombinant cell of claim 20, wherein one heterologousnucleic acid encodes an organic acid transporter having at least 60%amino acid identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO:
 5. 22. The recombinant cell of claim 1, further comprising agenetic disruption of one or more genes encoding a pyruvatedecarboxylase, a protein subunit of the pyruvate dehydrogenase complex,glycerol-3-phosphate dehydrogenase, NAD(P)H dehydrogenase, orcombinations thereof.
 23. The recombinant cell of claim 22, wherein onegenetic disruption is in a gene encoding a pyruvate decarboxylase havingat least 60% amino acid identity with a sequence selected from SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 24. The recombinantcell of claim 22, wherein one genetic disruption is in a gene encoding aglycerol-3-phosphate dehydrogenase having at least 60% amino acididentity with SEQ ID NO:
 10. 25. The recombinant cell of claim 22,wherein one genetic disruption is in a gene encoding an NAD(P)Hdehydrogenase having at least 60% amino acid identity with SEQ ID NO:11.
 26. A method of producing L-lactic acid comprising: culturing therecombinant cell of claim 1 under fermentation conditions suitable toproduce L-lactic acid, or a salt thereof. 27-34. (canceled)